Anti-ox40l antibodies

ABSTRACT

This invention relates to anti-OX40L antibodies and, in particular, to anti-OX40L antibodies and variants thereof that contain a Fc part derived from human origin and do not bind complement factor C 1 q. These antibodies have new and inventive properties causing a benefit for a patient suffering from inflammatory diseases.

This application claims benefit under 35 U.S.C. 119(e) of EuropeanPatent Applications No. EP 04022158.2 filed Sep. 17, 2004, and EP04030546.8 filed Dec. 24, 2004, which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to anti-OX40L antibodies and, inparticular, to anti-OX40L antibodies that do not bind complement factorC1q, pharmaceutical compositions and uses thereof. Preferably, theseantibodies are human or humanized antibodies.

BACKGROUND OF THE INVENTION

Human OX40L (gp34, SwissProt P23510) is expressed on activated B cellsand dendritic cells upon CD40/CD40L ligation, and on endothelial cellsin inflammatory tissues (Review: Weinberg, A. D., Trends Immunol. 23(2002) 102-109). It has first been isolated from HTLV-1 infected humanleukemic cells (immortalization of these T-cells by generation of anautokrine loop with OX40). OX40L and antibodies against are mentionede.g. in WO 95/12673; WO 95/21915; WO 99/15200; Baum, P. R., et al., EMBOJ. 13 (1994) 3992-4001; Imura, A., et al., Blood 89 (1997) 2951-2958;Imura, A., et al., J. Exp. Med. 183 (1996) 2185-2195; Kjaergaard, J., etal., J. Immunol. 167 (2001) 6669-6677; Lane, P., J. Exp. Med. 191 (2000)201-206; Mallett, S., and Barclay, A. N., Immunol. Today 12 (1991)220-223; Mallett, S., et al., EMBO J. 9 (1990) 1063-1068; Ndhlovu, L.C., et al., J. Immunol. 167 (2001) 2991-2999; Ohshima, Y., et al., J.Immunol. 159 (1997) 3838-3848; Rogers, P. R., et al., Immunity 15 (2001)445-455; Stüer, E., and Strober, W., J. Exp. Med. 183 (1996) 979-989;Stüber, E., et al., Gastroenterology 115 (1998) 1205-1215; Takahashi,Y., et al., J. Virol. 75 (2001) 6748-6757; Takasawa, N., et al., Jpn. J.Cancer Res. 92 (2001) 377-382; Taylor, L., and Schwarz, H., J. Immunol.Meth. 255 (2001) 67-72; Weinberg, A. D., et al., Nature Medicine 2(1996) 183-189; Weinberg, A. D., et al., Semin. Immunol. 10 (1998)471-480; Weinberg, A. D., Trends Immunol. 23 (2002) 102-109; Wu, T., etal., Transplant. Proc. 33 (2001) 217-218; Higgins, L. M., et al., J.Immunol. 162 (1999) 486-493; and Yoshioka, T., et al., Eur. J. Immunol.30 (2000) 2815-2823. Human OX40L is the ligand for human OX40 (CD 134)which is transiently expressed on activated CD4+ T cells. Engagement ofOX40 by its ligand leads to a costimulatory signal for T cellactivation. OX40/OX40L interaction is described to create abidirectional signal (Matsumura, Y., et al., J. Immunol. 163 (1999)3007-3011; Kotani, A., et al., Immunol. Lett. 84 (2002) 1-7). FurtherOX40/OX40L interaction mediate adhesion of activated T-cell toendothelial cells in inflammatory tissues. As OX40L is only transientlyexpressed on activated B cells, DC and endothelial cells, antibodies toOX40L should selectively block T cell activation and endothelial celladhesion during an inflammatory response but leave unactivated,peripheral T cells unaffected. Yoshioka, A., et al. (Eur. J. Immunol. 30(2000) 2815-2823) demonstrated the therapeutic potential of aneutralizing anti-mOX40L mAb in a mouse model for rheumatoid arthritis.Administration of it dramatically ameliorated the disease severity. Thisantibody showed similar activities in other related disease models, e.g.inflammatory skin disease, experimental autoimmune disease (EAE), GVHD,urine inflammatory bowel disease (Yoshioka, A., et al., Eur. J. Immunol30 (1999) 2815-2823; Salek-Ardakani, S., et al., J. Exp. Med. 198 (2003)315-324; Burgess, J. K., et al., J. Allergy Clin. Immunol. 113 (2004)683-689; Hoshino, A., et al., Eur. J. Immunol. 33 (2003) 861-869;Arestides, R. S., et al., Eur. J Immunol. 32 (2002) 2874-2880; Nohara,C., et al., J. Immunol. 166 (2001) 2108-2115; Weinberg, A. D., et al.,J. Immunol. 162 (1999) 1818-1826; Higgins, L. M., et al., J. Immunol.162 (1999) 486-493; Humphreys, I. R., et al., J. Exp. Med. 198 (2003)1237-1242; Akiba, H., et al., J. Exp. Med. 191 (2000) 375-380; Ishii,N., et al., Eur. J. Immunol. 33 (2003) 2372-2381; Blazar, B. R., et al.,Blood 101 (2003) 3741-3748; Tsukada, N., et al., Blood 95 (2000)2434-2439; Akiba, H., et al., Biochem. Biophys. Res. Commun. 251 (1998)131-136.

Antibodies against OX40L have been investigated for theiranti-inflammatory effects in various disease models (Sugamura, K., etal., Nat. Rev. Immunol. 4 (2004) 420-431). Tanaka, Y., et al, Int. J.Cancer 36, (1985) 549-555; Tozawa, H., et al., Int. J. Cancer 41 (1988)231-238; and Miura, S., et al., Mol. Cell. Biol. 11 (1991) 1313-1325describe mouse monoclonal antibodies named TARM-34 and TAG-34 that reactwith surface antigens of lines of human lymphocytes bearing a humanT-cell leukemia virus type-I (HTLV-I). TAG-34 antibody is commerciallyavailable from MBL International Corporation. TAG-34 binds also toOX40L.

SUMMARY OF THE INVENTION

The invention relates to an antibody, preferably a monoclonal antibody,characterized in that said antibody binds OX40L, contains a Fc part fromhuman origin and does not bind human complement factor C1q and/or humanFcγ receptor on NK cells.

The invention further relates to an antibody, preferably a monoclonalantibody characterized in that said antibody contains a Fc part fromhuman origin, binds to OX40L and to denatured OX40L (in a Western Blot)in an antibody concentration of 100 ng. This antibody binds to the sameOX40L polypeptide epitope as the epitope to which the monoclonalantibody LC.001 binds. Preferably this antibody does not bind humancomplement factor C1q and/or human Fcγ receptor on NK cells.

The antibody according to the invention is preferably characterized inthat non-binding of the antibody to complement factor C1q refers to anELISA assay measurement wherein the maximal binding (Bmax) of theantibody at a concentration of 10 μg/ml to C1q is 30% or lower,preferably 20% or lower compared to Bmax of antibody LC.001.

Preferably the antibody does not bind to human FcγRI, FcγRIIA and/orFcγRIIIA Especially preferred, the antibody does not bind to human Fcγreceptor on NK effector cells.

The antibody according to the invention is preferably characterized inthat non-binding of the antibody to Fcγ receptor on NK cells refers toassay wherein the maximal binding (Bmax) of the antibody at aconcentration of 20 μg/ml to NK cells is 20% or lower, preferably 10% orlower compared to Bmax of antibody LC.001.

The antibody according to the invention is preferably characterized inthat it does not bind to FcγRI. This means that the antibody ischaracterized by an EC₅₀ value which is five fold or more, preferablyseven fold or more, such as eight fold or more compared to the EC₅₀value of LC.001, when measured in an assay testing binding of theantibody in a concentration ranging from 0.078-10 μg/ml to a B-celllymphoma cell lacking FcγRIIA and FcγIIB, but expressing recombinantFcγRI.

The antibody has new and inventive properties causing a benefit for apatient in the need of a therapy with antibodies against OX40L,especially for a patient suffering from inflammatory disorders,especially from rheumatoid arthritis, allergic asthma, and GvHD intransplantation (see also Sugamura, K., et al., Nat. Rev. Immunol. 4(2004) 420-431).

The antibody according to the invention is preferably characterized asbeing an IgG4 antibody or an IgG1 antibody comprising at least one aminoacid mutation, preferably in the human Fc part, causing non-binding tocomplement factor C1q and/or non-binding to human Fcγ receptor on NKcells.

The antibody according to the invention is preferably a chimeric, humanor humanized antibody.

The antibody according to the invention is preferably characterized inthat it does not activate complement factor C3.

The antibody according to the invention is preferably characterized bybinding to OX40L with a K_(D) value of less than 10⁻⁸M (10⁻¹² to 10⁻⁸M), more preferably by a K_(D) range of 10⁻¹² to 10⁻⁹ M in a BIAcoreassay.

The antibody according to the invention is preferably characterized bybeing of human subclass IgG4. In a further preferred embodiment of theinvention, the antibody is characterized by being of any IgG class,preferably being IgG1 or IgG4, containing at least one mutation in E233,L234, L235, G236, D270, N297, E318, K320, K322, A327, A330, P331 and/orP329 (numbering according to EU index). Especially preferred are theIgG1 mutations PVA236, L234A/L235A and/or GLPSS331 as well as the IgG4mutation L235E. It is further preferred that the antibody of IgG4subclass contains the mutation S228P or the mutation S228P and L235E(Angal, S., et al., Mol. Immunol. 30 (1993) 105-108).

The antibody according to the invention therefore is preferably anantibody of human subclass IgG1, containing one or more mutation(s) fromPVA236, GLPSS331 and/or L234A/L235A (numbering according to EU index).

The antibody according to the invention preferably inhibits theinteraction of OX40L with OX40 in an ELISA using immobilized OX40L(preferably biotinylated OX40L immobilized on a streptavidine surface)at a coating concentration of 0.5 μg/ml with an IC₅₀ value of no morethan 4 nM. More preferred the IC₅₀ value is in the range of 1 to 4 nM.

The antibody according to the invention is preferably characterized inthat it does not elicit complement-dependent cytotoxicity (CDC).

The antibody according to the invention is preferably characterized inthat it does not elicit antibody-dependent cellular cytotoxicity (ADCC).

The antibody according to the invention is preferably characterized inthat said antibody binds OX40L and that the antibody comprises avariable region independently selected from the group consisting of

a) the light chain (V_(L)) variable domain defined by amino acidsequence SEQ ID NO:1 and the heavy chain (V_(H)) variable domain definedby SEQ ID NO:2;b) the light chain variable domain defined by amino acid sequence SEQ IDNO:3 and the heavy chain variable domain defined by SEQ ID NO:4;c) the light chain variable domain defined by amino acid sequence SEQ IDNO:5 and the heavy chain variable domain defined by SEQ ID NO:6;d) the light chain variable domain defined by amino acid sequence SEQ IDNO:7 and the heavy chain variable domain defined by SEQ ID NO:8;e) the light chain variable domain defined by amino acid sequence SEQ IDNO:9 and the heavy chain variable domain defined by SEQ ID NO:10;f) the light chain variable domain defined by amino acid sequence SEQ IDNO:11 or 16 and the heavy chain variable domain defined by SEQ ID NO:12

or an OX40L-binding fragment thereof.

The antibody according to the invention is preferably characterized inthat the human light chain variable region comprises an amino acidsequence independently selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11 and 16.

The antibody according to the invention is preferably characterized inthat the human heavy chain variable region comprises an amino acidsequence independently selected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10 and 12.

The CDR regions of the heavy and light chains are shown in SEQ ID NO:17-46.

The antibody according to the invention is preferably characterized inthat the antibody comprises the light chain variable domain defined byamino acid sequence SEQ ID NO:1 and the heavy chain variable domaindefined by SEQ ID NO:2.

The antibody according to the invention is preferably characterized inthat the human heavy chain constant region comprises an amino acidsequence independently selected from the group consisting of SEQ ID NO:14 and 15.

The antibody according to the invention is preferably characterized inthat the antibody comprises a κ-light chain constant region of SEQ IDNO: 13.

The antibody according to the invention is preferably characterized bycomprising a variable light chain and a variable heavy chain,characterized in that the variable heavy chain comprises CDR1, CDR2 andCDR3 characterized in that CDR3 is selected from SEQ ID NOs: 26-29. Itis especially preferred that CDR1 is selected from SEQ ID NOs:17-20,CDR2 is selected from SEQ ID NOs: 21-25 and CDR3 is selected from SEQ IDNOs: 26-29.

The antibody according to the invention is preferably characterized bycomprising a variable light chain and a variable heavy chain,characterized in that the variable light chain comprises CDR1, CDR2 andCDR3 characterized in that CDR3 is selected from SEQ ID NOs: 40-45. Itis especially preferred that CDR1 is selected from SEQ ID NOs: 30-34,CDR2 is selected from SEQ ID NOs: 35-39.and CDR3 is selected from SEQ IDNOs: 40-45.

The antibody according to the invention is preferably characterized bycomprising a variable heavy chain and a variable light chain,characterized in that the variable heavy chain comprises CDR1, CDR2 andCDR3 characterized in that CDR3 of the heavy chain is selected from SEQID NOs: 26-29 and CDR3 of the light chain is selected from SEQ ID NOs:40-45. It is especially preferred that the variable heavy chaincomprises CDR1 selected from SEQ ID NOs: 17-20, CDR2 selected from SEQID NOs: 21-25 and CDR3 selected from SEQ ID NOs: 26-29 and the variablelight chain comprises CDR1 selected from SEQ ID NOs: 30-34, CDR2selected from SEQ ID NOs: 35-39 and CDR3 selected from SEQ ID NOs:40-45.

All CDRs are selected independently from each other. Preferred are thecombinations of CDRs

A further embodiment of the invention is an antibody binding to OX40L,characterized in that it is produced by cell line hu-Mab<hOX40L>LC.001,hu-Mab<hOX40L>LC.005, hu-Mab<hOX40L>LC.010, hu-Mab<hOX40L>LC.019,hu-Mab<hOX40L>LC.029 or hu-Mab<hOX40L>LC.033.

The antibody according to the invention is preferably characterized inthat the antibody comprises CDRs independently selected from the groupconsisting of

a) the light chain (V_(L)) variable CDRs of amino acid sequence SEQ IDNO:1 and the heavy chain (V_(H)) variable CDRs of SEQ ID NO:2;b) the light chain variable CDRs of amino acid sequence SEQ ID NO:3′ andthe heavy chain variable CDRs of SEQ ID NO:4;c) the light chain variable CDRs of amino acid sequence SEQ ID NO:5 andthe heavy chain variable CDRs of SEQ ID NO:6;d) the light chain variable CDRs of amino acid sequence SEQ ID NO:7 andthe heavy chain variable CDRs of SEQ ID NO:8;e) the light chain variable CDRs of amino acid sequence SEQ ID NO:9 andthe heavy chain variable CDRs of SEQ ID NO:10;f) the light chain variable CDRs of amino acid sequence SEQ ID NO:11 or16 and the heavy chain variable CDRs of SEQ ID NO:12 or an OX40L-bindingfragment thereof.

A further embodiment of the invention is a nucleic acid moleculeencoding an antibody molecule, a variable chain or a CDR domain thereofaccording to the invention.

CDRs on each chain are separated by framework amino acids.

In a preferred embodiment of the invention the antibody is a Fab,F(ab′)₂ or a single-chain fragment.

A further embodiment of the invention is a vector comprising the nucleicacid molecule according to the invention.

A further embodiment of the invention is a host cell comprising thevector according to the invention.

A further embodiment of the invention is a method for the preparation ofan antibody according to the invention comprising culturing the hostcell according to the invention under conditions that allow synthesis ofsaid antibody molecule and recovering said antibody molecule from saidculture.

A further embodiment of the invention is a composition, preferably apharmaceutical or a diagnostic composition of the antibody according tothe invention.

A further embodiment of the invention is a pharmaceutical compositioncomprising an antibody according to the invention and at least onepharmaceutically acceptable excipient.

A further embodiment of the invention is a method for the treatment of apatient in need of therapy, characterized by administering to thepatient a therapeutically effective amount of an antibody according tothe invention.

A further embodiment of the invention is the use of an antibodyaccording to the invention for therapy, preferably for the treatment ofinflammatory diseases, especially for the treatment and/or prevention ofrheumatoid arthritis, asthma and GvHD (graft versus host disease).

A further embodiment of the invention is the use of an antibodyaccording to the invention for the preparation of a medicament for theprophylaxis and/or treatment of inflammatory disorders, preferably forthe treatment of rheumatoid arthritis, asthma and GvHD.

A further embodiment of the invention is a diagnostic kit comprising anantibody according to the invention, a nucleic acid molecule accordingto the invention, a vector according to the invention or a host cellaccording to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows “binding ELISA” for TAG-34, LC.001, LC.005, LC.010,LC.019, LC.029, LC.033.

FIG. 1 b shows “blocking ELISA” +IC50 data for TAG-34, LC.001, LC.005,LC.010, LC.019, LC.029, LC.033.

FIG. 2 shows “blocking FACS” for TAG-34, LC.001, LC.005.

FIG. 3 shows “NFkB-assay” for TAG-34, LC.001, LC.019 and LC.024 (nonbinding antibody).

FIGS. 4 and 5 show “T-cell activation assay” and IC50-values for TAG-34,LC.001 and LC.005 (FIG. 4: IL-2 release, FIG. 5: inhibition).

FIG. 6 shows “TT-assay” data for TAG-34, LC.001 and LC.033.

FIG. 7 shows the cross-reactivity of the antibodies of the inventionwith mouse OX40L. A) control for hOX40L expression on transfected and WTcells, B) binding of the antibodies to hOX40L expressing K562 cells, C)control for mOX40L expression on transfected and WT cells, D) binding ofthe antibodies to mOX40L expressing K562 cells, and E) binding of theantibodies to WT K562 cells (n=3).

FIG. 8 shows the ability of the antibodies of the invention to bind C1q(n=3).

FIG. 9 shows the ability of the antibodies of the invention to activateC3c (n=3).

FIG. 10 shows the ability of the antibodies of the invention to bind toFcγRI (n=4), FcγRIIa (n=4) and FcγRIIb (n=4).

FIG. 11 shows the ability of the antibodies of the invention to bind toFcγRIIIa (CD16) on NK cells (Mean±SEM of 6 donors).

FIG. 12 Western Blot; lanes 1, 4, 7: marker; lanes 2, 5, 8: 100 ngOX40L; lanes 3, 6, 9: 40 ng OX40L.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 kappa light chain, variable region of LC.001

SEQ ID NO:2 γ heavy chain variable region of LC.001

SEQ ID NO:3 kappa light chain, variable region of LC.005

SEQ ID NO:4 γ heavy chain variable region of LC.005

SEQ ID NO:5 kappa light chain, variable region of LC.010

SEQ ID NO:6 γ heavy chain variable region of LC.010

SEQ ID NO:7 kappa light chain, variable region of LC.029

SEQ ID NO:8 γ heavy chain variable region of LC.029

SEQ ID NO:9 kappa light chain, variable region of LC.019

SEQ ID NO:10 γ heavy chain variable region of LC.019

SEQ ID NO:11 kappa light chain, variable region of LC.033

SEQ ID NO:12 γ heavy chain variable region of LC.033

SEQ ID NO:13 kappa light chain constant region

SEQ ID NO:14 γ1 heavy chain constant region

SEQ ID NO:15 γ4 heavy chain constant region

SEQ ID NO:16 kappa light chain, mutant variable region of LC.033

SEQ ID NO:17-45 CDR sequences

DETAILED DESCRIPTION OF THE INVENTION

The term “OX40L” refers to a type II membrane protein belonging to theTNF-ligand family. Further names are ACT-4 receptor, CD134L, gp34 orTNF4_Human. It has a molecular weight of 34 KDa and is stored inSwissProt with the accession number P23510.

The term “OX40” confers to the receptor which binds to OX40L. It is atype I membrane protein belonging to the TNF receptor family. Furthernames are ACT-4, OX40L receptor, CD134 antigen, ACT35 antigen,TNR4_Human. It has a molecular weight of 50 kDa and is stored inSwissProt with the accession number P43489.

The term “antibody” encompasses the various forms of antibodies,preferably monoclonal antibodies including but not being limited towhole antibodies, antibody fragments, human antibodies, chimericantibodies, humanized antibodies and genetically engineered antibodies(variant or mutant antibodies) as long as the characteristic propertiesaccording to the invention are retained. Especially preferred are humanor humanized monoclonal antibodies, especially as recombinant humanantibodies.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are preferred. Other preferred forms of “chimeric antibodies”encompassed by the present invention are those in which the constantregion has been modified or changed from that of the original antibodyto generate the properties according to the invention, especially inregard to C1q binding and/or Fc receptor (FcR) binding. Such chimericantibodies are also referred to as “class-switched antibodies.” Chimericantibodies are the product of expressed immunoglobulin genes comprisingDNA segments encoding immunoglobulin variable regions and DNA segmentsencoding immunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos.5,202,238 and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric and bifunctional antibodies. Other forms of“humanized antibodies” encompassed by the present invention are those inwhich the constant region has been additionally modified or changed fromthat of the original antibody to generate the properties according tothe invention, especially in regard to C1q binding and/or Fc receptor(FcR) binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. andBoerner et al. are also available for the preparation of humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J.Immunol. 147 (1991) 86-95). As already mentioned for chimeric andhumanized antibodies according to the invention the term “humanantibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation). In addition the inventioncomprises monoclonal human antibodies against OX40L which bind to C1qand/or FcR. Such human antibodies are characterized by a highselectivity for for human OX40L vs. mouse OX40L (>30 fold lower bindingto mouse OX40L than to human OX40L) and do not show unspecific bindingto TNFα or CD40L up to a concentration of 500 nM. Such antibodies areuseful for generation of antibodies which do not bind C1q and/or FcR.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NSO or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germ line V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germ line repertoire in vivo.

The “variable region” (variable region of a light chain (V_(L)),variable region of a heavy chain (V_(H))) as used herein denotes each ofthe pair of light and heavy chains which is involved directly in bindingthe antibody to the antigen. The domains of variable human light andheavy chains have the same general structure and each domain comprisesfour framework (FR) regions whose sequences are widely conserved,connected by three “hypervariable regions” (or complementaritydetermining regions, CDRs). The framework regions adopt a β-sheetconformation and the CDRs may form loops connecting the β-sheetstructure. The CDRs in each chain are held in their three-dimensionalstructure by the framework regions and form together with the CDRs fromthe other chain the antigen binding site. The antibody heavy and lightchain CDR3 regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The terms “hypervariable region” or “antigen-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. CDRs on each chain are separated by such framework aminoacids. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

The term “nucleic acid or nucleic acid molecule”, as used herein, isintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The “constant domains” are not involved directly in binding an antibodyto an antigen, but exhibit various effector functions. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, ε, γ, and μ, respectively. The antibodiesaccording to the invention are preferably of IgG type.

The Fc part of an antibody is directly involved in complementactivation, C1q binding, C3 activation and Fc receptor binding. Whilethe influence of an antibody on the complement system is dependent oncertain conditions, binding to C1q is caused by defined binding sites inthe Fc part. Such binding sites are known in the state of the art anddescribed e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981)2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16 (1979)907-917; Burton, D. R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E. E., et al.,J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75(2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324;and EP 0 307 434. Such binding sites are e.g. L234, L235, D270, N297,E318, K320, K322, P331 and P329 (numbering according to EU index ofKabat, see below). Antibodies of subclass IgG1, IgG2 and IgG3 usuallyshow complement activation, C1q binding and C3 activation, whereas IgG4do not activate the complement system, do not bind C1q and do notactivate C3. As used herein the term “Fc part derived from human origin”denotes a Fc part which is either a Fc part of a human antibody of thesubclass IgG4 or a Fc part of a human antibody of the subclass IgG1,IgG2 or IgG3 which is modified in such a way that no C1q binding, C3activation and/or FcR binding as defined below can be detected. A “Fcpart of an antibody” is a term well known to the skilled artisan anddefined on the basis of papain cleavage of antibodies. The antibodiesaccording to the invention contain as Fc part, preferably a Fc partderived from human origin and preferably all other parts of the humanconstant regions. Preferably the Fc part is a human Fc part andespecially preferred either from human IgG4 subclass, preferably mutatedin the hinge region (e.g. S228P and/or L235E) or a mutated Fc part fromhuman IgG1 subclass. Mostly preferred are Fc parts comprising a heavychain constant regions selected from the regions shown in SEQ ID NO: 14and 15, SEQ ID NO: 14 with mutations L234A and L235A or SEQ ID NO:15with mutation S228P or mutations S228P and L235E.

The present invention refers to an antibody that binds OX40L and doesnot bind complement factor C1q, and/or Fc receptor. In a preferredembodiment of the invention, these antibodies do not elicit thecomplement dependent cytotoxicity (CDC) and/or antibody-dependentcellular cytotoxicity (ADCC). Preferably, this antibody is characterizedin that it binds OX40L, contains a Fc part derived from human origin anddoes not bind complement factor C1q. More preferably, this antibody is ahuman or humanized monoclonal antibody.

The effector functions mediated by the Fc part of the antibody Fc regionrefer to effector functions that operate after the binding of anantibody to an antigen (these functions involve the activation of thecomplement cascade and/or cell activation by a Fc receptor).

The function of the complement cascade can be assessed by the CHSOassay. Sheep red cells sensitized with anti-red cell antibodies (EA) areadded to test serum to activate the classical pathway resulting inhaemolysis. The volume of serum needed to lyse 50% of the red cellsdetermines the CHSO unit. The AP-CHSO measures the alternative and theterminal pathways. The procedure is similar except that rabbit red cellsare used. The alternative pathway is activated upon addition of testserum.

C1q and two serine proteases, C1r and C1s, form the complex C1, thefirst component of the complement dependent cytotoxicity (CDC) pathway.To activate the complement cascade C1q binds to at least two moleculesof IgG1 or one molecule of IgM, attached to the antigenic target (Ward,E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, D. R.,described (Mol. Immunol. 22 (1985) 161-206) that the heavy chain regioncomprising amino acid residues 318 to 337 is being involved incomplement fixation. Duncan, A. R., and Winter, G. (Nature 332 (1988)738-740), using site directed mutagenesis, reported that Glu318, Lys320and Lys322 form the binding site to C1q. The role of Glu318, Lys320 andLys 322 residues in the binding of C1q was confirmed by the ability of ashort synthetic peptide containing these residues to inhibit complementmediated lysis.

The term “complement-dependent cytotoxicity (CDC)” refers to lysis ofOX40L expressing human endothelial cells by the antibody according tothe invention in the presence of complement. CDC is measured preferablyby the treatment of OX40L expressing human endothelial cells with anantibody according to the invention in the presence of complement. Thecells are preferably labeled with calcein. CDC is found if the antibodyinduces lysis of 20% or more of the target cells at a concentration of30 μg/ml. The inventors have found that for the properties of theantibodies according to the invention reduced binding to the complementfactor C1q in an ELISA assay is essential. In such an assay in principlean ELISA plate is coated with concentration ranges of the antibody, towhich purified human C1q or human serum is added. C1q binding isdetected by an antibody directed against C1q followed by aperoxidase-labeled conjugate. Detection of binding (maximal bindingBmax) is measured as optical density at 405 nm (OD₄₀₅) for peroxidasesubstrate ABTS® (2,2′-azino-di-[3-ethylbenz-thiazoline-6-sulfonate (6)].Accordingly the present invention refers to an antibody, characterizedin that non-binding of the antibody to complement factor C1q refers tosuch an ELISA assay measurement wherein the maximal binding (Bmax) ofC1q to an antibody according to the invention at a concentration of 10μg/ml of the antibody is 20% or lower of Bmax observed with antibodyLC.001, preferably 10% or lower.

It is further preferred, that an antibody according to the inventionshows a reduced activation of complement factor C3 in an ELISA assay.The assay is performed in the same manner as the C1q assay. In such anassay in principle an ELISA plate is coated with concentration ranges ofthe antibody, to which human serum is added. C3 binding is detected byan antibody directed against C3 followed by a peroxidase-labeledconjugate. Detection of binding (maximal binding Bmax) is measured asoptical density at 405 nm (OD₄₀₅) for peroxidase substrate ABTS®.Accordingly the present invention refers to an antibody, characterizedin that non-binding of the antibody to complement factor C3 refers tosuch an ELISA assay measurement wherein the maximal binding (Bmax) of C3to the antibody at a concentration of 10 μg/ml of the antibody is 10% ofBmax of antibody LC.001 preferably 5% or lower.

The term “antibody-dependent cellular cytotoxicity (ADCC)” is a functionmediated by Fc receptor binding and refers to lysis of OX40L expressingtarget cells by an antibody according to the invention in the presenceof effector cells. ADCC is measured preferably by the treatment of apreparation of OX40L expressing erythroid cells (e.g. K562 cellsexpressing recombinant human OX40L) with an antibody according to theinvention in the presence of effector cells such as freshly isolatedPBMC (peripheral blood mononuclear cells) or purified effector cellsfrom buffy coats, like monocytes or NK (natural killer) cells. Targetcells are labeled with ⁵¹Cr and subsequently incubated with theantibodies. The labeled cells are incubated with effector cells and thesupernatant is analyzed for released ⁵¹Cr. Controls include theincubation of the target endothelial cells with effector cells butwithout the antibody. The capacity of the antibodies to induce theinitial steps mediating ADCC was investigated by measuring their bindingto Fcγ receptors expressing cells, such as cells, recombinantlyexpressing FcγRI and/or FcγRIIA or NK cells (expressing essentiallyFcγRIIIA). Preferably binding to FcγR on NK cells is measured.

Fc receptor binding effector functions can be mediated by theinteraction of the Fc region of an antibody with Fc receptors (FcRs),which are specialized cell surface receptors on hematopoietic cells. Fcreceptors belong to the immunoglobulin superfamily, and have been shownto mediate both the removal of antibody-coated pathogens by phagocytosisof immune complexes, and the lysis of erythrocytes and various othercellular targets (e.g. tumor cells) coated with the correspondingantibody, via antibody dependent cell mediated cytotoxicity (ADCC). Vande Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991)511-524). FcRs are defined by their specificity for immunoglobulinisotypes; Fc receptors for IgG antibodies are referred to as FcγR, forIgE as FcεR, for IgA as FcαR and so on. Fc receptor binding is describede.g. in Ravetch, J. V., and Kinet, J. P., Annu. Rev. Immunol. 9 (1991)457-492; Capel, P. J., et al., Immunomethods 4 (1994) 25-34; de Haas,M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E.,et al., Ann. Hematol. 76 (1998) 231-248.

Cross-linking of receptors for the Fc domain of IgG antibodies (FcγR)triggers a wide variety of effector functions including phagocytosis,antibody-dependent cellular cytotoxicity, and release of inflammatorymediators, as well as immune complex clearance and regulation ofantibody production. In humans, three classes of FcγR have beencharacterized, which are:

-   -   FcγRI (CD64) binds monomeric IgG with high affinity and is        expressed on macrophages, monocytes, neutrophils and        eosinophils. Modification in IgG of at least one of E233-G236,        P238; D265, N297, A327 and P329 reduce binding to FcγRI. IgG2        residues at positions 233-236, substituted into IgG1 and IgG4,        reduced binding to FcγRI by 10³-fold and eliminated the human        monocyte response to antibody-sensitized red blood cells        (Armour, K. L., et al., Eur. J. Immunol. 29 (1999) 2613-2624).    -   FcγRII (CD32) binds complexed IgG with medium to low affinity        and is widely expressed. These receptors can be divided into two        important types, FcγRIIA and FcγRIIB. FcγRIIA is found on many        cells involved in killing (e.g. macrophages, monocytes,        neutrophils) and seems able to activate the killing process.        FcγRIIB seems to play a role in inhibitory processes and is        found on B-cells, macrophages and on mast cells and eosinophils.        On B-cells it seems to function to suppress further        immunoglobulin production and isotype switching to say for        example the IgE class. On macrophages, FcγRIIB acts to inhibit        phagocytosis as mediated through FcγRIIA. On eosinophils and        mast cells the b form may help to suppress activation of these        cells through IgE binding to its separate receptor. Reduced        binding for FcγRIIA is found e.g. for IgG mutation of at least        one of E233-G236, P238, D265, N297, A327, P329, D270, Q295,        A327, 8292 and K414.    -   FcγRIII (CD16) binds IgG with medium to low affinity and exists        as two types. FcγRIIIA is found on NK cells, macrophages,        eosinophils and some monocytes and T cells and mediates ADCC.        FcγRIIIB is highly expressed on neutrophils. Reduced binding to        FcγRIIIA is found e.g. for mutation of at least one of        E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239,        E269, E293, Y296, V303, A327, K338 and D376.

Mapping of the binding sites on human IgG1 for Fc receptors, the abovementioned mutation sites and methods for measuring binding to FcγRI andFcγRIIA are described in Shields, R. L., et al., J. Biol. Chem. 276(2001) 6591-6604.

The term “Fc receptor” when used herein, refer to activation receptorscharacterized by the presence of a cytoplasmatic ITAM sequenceassociated with the receptor (see e.g. Ravetch, J. V., and Bolland, S.,Annu. Rev. Immunol. 19 (2001) 275-290). Such receptors are FcγRI,FcγRIIA and FcγRIIIA. The antibodies according to the inventionpreferably show a reduced binding to Fcγ receptors, preferably toFcγIIIA. Preferably the term “no binding of FcγR” means that in anantibody concentration of 10 μg/ml the binding of an antibody accordingto the invention to NK cells is 10% or less of the binding found forantibody LC.001.

While IgG4 shows reduced FcR binding, antibodies of other IgG subclassesshow strong binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fccarbohydrate), Pro329 and 234, 235, 236 and 237 11e253, Ser254, Lys288,Thr307, Gln311, Asn434, and His435 are residues which provide if alteredalso reduce FcR binding (Shields, R. L., et al. J. Biol. Chem. 276(2001) 6591-6604; Lund, J., et al. FASEB J. 9 (1995) 115-119; Morgan,A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434). Preferablyan antibody according to the invention of IgG1 or IgG2 subclasscomprises mutation PVA236, GLPSS331 and/or L234A/L235A. An antibodyaccording to the invention of IgG4 subclass comprises preferablymutation L235E. Further preferred IgG4 mutations are S228P or L235E andS228P (cf. table 1).

The term “binding to OX40L” as used herein means the binding of theantibody to human OX40L in a BIAcore assay (Pharmacia Biosensor AB,Uppsala, Sweden). For further confirmation, binding to OX40L can also bedetermined in an ELISA in which purified OX40L is coated to microtiterplates, or in a FACS-assay in which direct or indirect labeled antibodyis bound to K562 cells expressing OX40L.

In the BlAcore assay the antibody is bound to a surface and binding ofOX40L is measured by Surface Plasmon Resonance (SPR). The affinity ofthe binding is defined by the terms ka (rate constant for theassociation of the antibody to the antigen), kd (rate constant for thedissociation), and K_(D) (kd/ka). The antibodies according to theinvention show a K_(D) of 10⁻⁸ or less, preferably of about 10⁻¹² to10⁻⁹ M (see examples). Accordingly, the present invention refers to anantibody as described above, wherein the antibody bind to OX40L with aK_(D) value of less than 10⁻⁸ M in a BlAcore assay, preferably whereinthe K_(D) range is 10⁻¹² to 10⁻⁹ M.

In the OX40L-specific binding ELISA, OX40L is coated onto microtiterplates and the binding of the antibody to OX40L is detected with aHRP-conjugated anti-human IgG and the usual steps of an ELISA. The EC₅₀values in this assay are preferably in the range between 3 nM and 8 nM.

The term “inhibiting the binding of OX40 to OX40L” as used herein refersto the binding of the antibody described in the present invention tohuman OX40L, thereby inhibiting the OX40/OX40L interaction, and therebyinhibiting the OX40L induced signal transduction.

The antibodies of the present invention inhibit hOX40L/OX40 interactionpreferably

i) at the in vitro level shown by an ELISA assay by blocking theinteraction of biotinylated, immobilized OX40L with soluble OX40 by theantibody at a (solid phase) coating concentration of 0.5 μg/mlbiotinylated OX40L with an IC₅₀ value in the range of 1 nM-4 nM,ii) at the in vitro level shown by a Biacore assay by blocking theinteraction of immobilized OX40 with soluble OX40L (10 nM, preferably ashOX40L-His) by the antibody at an antibody concentration of 0.78-100 nMwith an IC₅₀ value in the range of 1 nM-10 nM,iii) on the cellular level shown by a FACS-assay in which the antibodyblocks the interaction of K562 cells expressing OX40L (K562_OX40L) in aconcentration of 2×10⁵ cells/sample with OX40 with an IC₅₀ value in therange of 4-20 nM,iv) by an OX40-signal transduction assay in which the antibody blocksthe OX40 signal transduction induced by K562_OX40L, into 3×10⁴HeLa-cells expressing OX40 per sample, which results in a blocking ofNFKB activation with an IC₅₀ value in the range of 1-5 nM,v) by a T-cell activation assay, in which the antibody blocks OX40Linduced T-cell activation by K562_OX40L at a concentration of 1.5×10⁵cells/sample and a PHA concentration of 0.75 μg/ml with an IC50 value inthe range of 1 nM to 10 nM, and/orvi) by a T-cell activation assay, in which the antibody blocks OX40Linduced T cell activation by activated B cells or dendritic cells(Tetanus assay) at an antibody concentration of 10 μg/ml, inhibition of40%-60% was obtained.

Antibodies showing in an ELISA assay inhibition by blocking theinteraction of immobilized OX40L with soluble OX40 at a coatingconcentration of 0.5 μg/ml OX40L with an IC50 value in the range of 1nM-4 nM are preferred.

Accordingly, a further preferred embodiment of the present inventionrefer to an antibody, characterized in that said antibody, therebyinhibiting the OX40/OX40L interaction, and thereby inhibiting the OX40Linduced signal transduction.

It is further preferred, that an antibody according to the inventiondoes not show unspecific binding to TNFalpha and CD40L up to aconcentration of 500 nM of TNFalpha or CD40L.

It is further preferred, that an antibody according to the inventionshow at least 30 fold lower binding to mouse OX40L compared to humanOX40L.

It is further preferred, that an antibody according to the invention ina concentration of 10 μg/ml do not induce downregulation of OX40Lexpression on HUVEC cells.

In a further preferred embodiment, the antibodies of the presentinvention are characterized in that they comprise a variable domaincombination independently selected from the group consisting ofcombinations

a) the light chain variable domain of antibody LC.001 defined by aminoacid sequence SEQ ID NO:1 and the heavy chain variable domain ofantibody LC.001 defined by SEQ ID NO:2;b) the light chain variable domain of antibody LC.005 defined by aminoacid sequence SEQ ID NO:3 and the heavy chain variable domain of theantibody LC.005 defined by SEQ ID NO:4;c) the light chain variable domain of antibody LC.010 defined by aminoacid sequence SEQ ID NO:5 and the heavy chain variable domain of theantibody LC.010 defined by SEQ ID NO:6;d) the light chain variable domain of antibody LC.029 defined by aminoacid sequence SEQ ID NO:7 and the heavy chain variable domain of theantibody LC.029 defined by SEQ ID NO:8;e) the light chain variable domain of antibody LC.019 defined by aminoacid sequence SEQ ID NO:9 and the heavy chain variable domain of theantibody LC.019 defined by SEQ ID NO:10;f) the light chain variable domain of antibody LC.033 defined by aminoacid sequence SEQ ID NO:11 OR 16 and the heavy chain variable domain ofthe antibody LC.033 defined by SEQ ID NO:12.

In an further preferred embodiment, the antibodies of the presentinvention are characterized in that they comprise a constant regionindependently selected from the group consisting of

g) the light/kappa chain defined by sequence SEQ ID NO:13;h) the heavy/gamma chain of the IgG1 isotype SEQ ID NO:14 with one ormore mutations selected from L234A and L235A, PVA236 or GLPSS331;i) the heavy/gamma chain of the IgG4 isotype SEQ ID NO:15;j) the heavy/gamma chain of the IgG4 isotype SEQ ID NO:15 with themutation S228P or mutations S228P and L235E. Further preferred are allcombinations of each variable antibody domain combination a)-f) togetherwith a gamma chain h), i) or j) and preferably with a kappa chain g).

Especially preferred are antibodies comprising the variable chains ofantibodies LC.001, LC.005, LC.010, LC.019, LC.029 or LC.033 each withkappa chain defined by sequence SEQ ID NO:13 and the heavy/gamma chainof the IgG1 isotype SEQ ID NO:14 with the mutations L234A and L235A,antibodies comprising the variable chains of antibodies LC.001, LC.005,LC.010, LC.019, LC.029 or LC.033 each with kappa chain defined bysequence SEQ ID NO:13 and the heavy/gamma chain of the IgG4 isotype SEQID NO:15, antibodies comprising the variable chains of antibodiesLC.001, LC.005, LC.010, LC.019, LC.029 or LC.033 each with kappa chaindefined by sequence SEQ ID NO:13 and the heavy/gamma chain of the IgG4isotype SEQ ID NO:15 with the mutation S228P.

Preferably, the antibodies comprise the light chain variable CDR ofamino acid sequence SEQ ID NO:1 and the heavy chain variable CDR of SEQID NO:2.

The preferred antibodies are characterized in that the antibodies are ofhuman IgG4 subclass or of another human subclass (preferably IgG1)comprising at least one amino acid mutation causing non-binding tocomplement factor C1q and/or loss of FCR binding. Such preferred variantantibodies comprise for example the amino acid sequence SEQ ID NO: 14with the mutations L234A and L235A or SEQ ID NO:15 with or withoutmutation S228P.

Preferred antibodies according to the invention are antibodies definedas IgG1v1 (PVA-236; GLPSS331 as specified by E233P; L234V; L235A; deltaG236; A327G; A330S; P331S), IgG1v2 (L234A; L235A) and IgG4v1 (S228P;L235E) and IgG4× (S228P).

Hybridoma cell line hu-Mab<hOX40L>LC.001 according to the invention wasdeposited, under the Budapest Treaty on the international recognition ofthe deposit of microorganisms for the purposes of patent procedure, withDeutsche Sammlung von Mikroorganismen und Zelikulturen GmbH (DSMZ),Germany on 27, Jul. 2004 under Deposition No. DSM ACC 2672.

Hybridoma cell lines hu-Mab<hOX40L>LC.005 (DSM ACC 2685),hu-Mab<hOX40L>LC.010 (DSM ACC 2686), hu-Mab<hOX40L>LC.019,hu-Mab<hOX40L>LC.029 (DSM ACC 2688) and hu-Mab<hOX40L>LC.033 (DSM ACC2689) according to the invention were deposited, under the BudapestTreaty on the international recognition of the deposit of microorganismsfor the purposes of patent procedure, with Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSMZ), Germany on 2, Sep. 2004.

The antibodies obtainable from said cell lines are preferred embodimentsof the invention and especially useful as intermediate substances forthe generation of antibodies according to the invention not bindingcomplement factor C1q and/or not binding to human Fcγ receptor.

Further preferred embodiments of the invention are isolated anti-OX40Lantibodies which bind to OX40L and bind to the same OX40L-epitope towhich also monoclonal antibodies LC.005, LC.010 or LC.029 produced bythe hybridoma cell lines deposited bind.

A further embodiment of the invention is a method for the production ofan antibody against OX40L which do not bind human complement factor C1qand/or human Fcγ receptor characterized in that the sequence of anucleic acid encoding the heavy chain of an antibody binding to OX40Lwith a KD value of less than 10⁻⁸M is modified in such a manner thatsaid modified antibody does not bind complement factor C1q and/or humanFcγ receptor on NK cells, said modified nucleic acid and the nucleicacid encoding the light chain of said antibody are inserted into anexpression vector, said vector is inserted in a prokaryotic oreukariotic host cell, the encoded protein is expressed and recoveredfrom the host cell or the supernant.

A further embodiment of the invention is a method for the production ofan antibody according to the invention not binding complement factor C1qand/or not binding to human Fcγ receptor characterized in that anantibody obtainable from one of said cell lines is modified by “classswitching”, i.e. change or mutation of the Fc part (e.g. from IgG1 toIgG4 and/or IgG1/IgG4 mutation) preferably defined as IgG1v1 (PVA-236;GLPSS331 as specified by E233P; L234V; L235A; delta G236; A327G; A330S;P331S), IgG1v2 (L234A; L235A) and IgG4v1 (S228P; L235E) and IgG4×(S228P).

In a further preferred embodiment, these antibodies also compriseantibody fragments selected from the group consisting of Fab, F(ab′)₂and single-chain fragments.

A “variant” anti-OX40L antibody, refers therefore herein to a moleculewhich differs in amino acid sequence from a “parent” anti-OX40L antibodyamino acid sequence by virtue of addition, deletion and/or substitutionof one or more amino acid residue(s) in the parent antibody sequence. Inthe preferred embodiment, the variant comprises one or more amino acidsubstitution(s) in one or more constant or variable region(s) of theparent antibody, preferably in the constant region. For example, thevariant may comprise at least one, e.g. from about one to about ten, andpreferably from about two to about five, substitutions in one or morevariable regions of the parent antibody. Ordinarily, the variant willhave an amino acid sequence having at least 90% amino acid sequenceidentity with the parent antibody constant and/or variable domainsequences, more preferably at least 95%, and most preferably at least99%.

Identity or homology with respect to this sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence shall beconstrued as affecting sequence identity or homology. The variantretains the ability to bind human OX40L and preferably has properties,which are superior to those of the parent antibody. For example, thevariant may have reduced side effects during treatment of rheumatoidarthritis and asthma as the OX40L is not only transiently expressed onB-cells, Dendritic cells and macrophages, but also on endothelial cells(Kotani, A., et al., Immunol. Lett. 84 (2002) 1-7), airway smooth musclecells (=ASM) (Burgess, J. K., J. Allergy Clin. Immunol 113 (2004)683-689) and microglial cells (Weinberg, A. D., et al., J. Immunol. 162(1999) 1818-1826). Binding of the antibodies against OX40L toendothelial cells, ASM and Microglial cells can result in cell damageand of endothelial cells resulting in vascular leakage, of ASM cellsresulting in lung destruction, of microglial cells resulting in damagesof the microglia.

The “parent” antibody herein is one, which is encoded by an amino acidsequence used for the preparation of the variant. Preferably, the parentantibody has a human framework region and, if present, has humanantibody constant region(s). For example, the parent antibody may be ahumanized or human antibody, preferably of IgG1 type.

The antibodies according to the invention include, in addition, suchantibodies having “conservative sequence modifications”, nucleotide andamino acid sequence modifications, which do not affect or alter theabove-mentioned characteristics of the antibody according to theinvention. Modifications can be introduced by standard techniques knownin the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-OX40L antibodycan be preferably replaced with another amino acid residue from the sameside chain family.

Amino acid substitutions can be performed by mutagenesis based uponmolecular modeling as described by Riechmann, L., et al., Nature 332(1988) 323-327 and Queen, C., et al., Proc. Natl. Acad. Sci. USA 86(1989) 10029-10033.

The invention further comprises a method for the production of anantibody, characterized in that the sequence of a first nucleic acidencoding the heavy chain of an antibody binding to OX40L with a KD valueof less than 10⁻⁸ M is modified in such a manner that said modifiedantibody do not bind complement factor C1q and/or human Fcγ receptor onNK cells, said modified first nucleic acid and a second nucleic acidencoding the light chain of said antibody are inserted into anexpression vector, said vector is inserted in a prokaryotic oreukariotic host cell, culturing said host cell under conditions thatallow synthesis of said antibody and recovering said antibody from saidculture.

The invention further comprises a method for the production of anantibody according to the invention and comprising a Fc part derivedfrom human origin, said method comprising the steps of a) transforming ahost cell with a first nucleic acid sequence encoding a light chain of aparent human antibody according to the invention and a second DNAsequence encoding a heavy chain of said parent human antibody whereinthe Fc part is modified in that said Fc part does not bind complementfactor C1q and/or Fc receptor; b) expressing said first and second DNAsequence so that said antibody heavy and light chains are produced andc) recovering said antibody from the host cell or host cell culture.

The present invention also comprises nucleic acid molecules encoding anantibody mentioned above, the corresponding vectors comprising thesenucleic acids and the corresponding host cell for these vectors. Theinvention encompasses a method for the preparation of the antibodiescomprising culturing the corresponding host cells under conditions thatallow synthesis of said antibody molecules and recovering saidantibodies from said culture, e.g. by expressing a nucleic acid encodinga heavy chain and a nucleic acid encoding a light chain in a prokaryoticor eukaryotic host cell and recovering said polypeptide from said cell.

Diagnostic and therapeutic uses for the antibody are contemplated. Inone diagnostic application, the invention provides a method fordetermining the presence of the OX40L protein comprising exposing asample suspected of containing OX40L to the anti-OX40L antibody anddetermining binding of the antibody to the sample. The OX40L protein maybe inserted into the cell membrane of OX40L-expressing cells by itstransmembrane domain or may occur as soluble extracellular domain inbody fluids released by mechanisms like shedding or proteolytic release.For this use, the invention provides a kit comprising the antibody andinstructions for using the antibody to detect the OX40L protein.

The antibodies of the present invention are useful for prevention and/ortreatment of inflammatory diseases in a mammal, preferably a patientsuspected of having or suffering of such a disease. Such diseasesinclude allergic reactions such as asthma. Other applications are thetreatment of autoimmune diseases including rheumatoid arthritis.

The invention further provides a method for treating a mammal sufferingfrom the above mentioned inflammatory disorders, especially from asthmaand rheumatoid arthritis.

Preferably the antibodies of the present invention can be used for thetreatment of severe persistent asthma in patients whose symptoms are notadequately controlled with inhaled corticosteroids. The patientpopulation includes adults and adolescents (12 years of age and older)with inadequately controlled severe persistent asthma. The antibody willbe delivered preferably subcutaneously once or twice a month. Mainendpoint will be preferably decrease in acute exacerbations. Otherendpoints include peak flow, daytime asthma symptoms, nocturnalawakenings, quality of life, emergency room visits, asthma free days,beta-2 agonist use, steroid reduction or tapering and effect onhyper-responsiveness.

It is further preferred to use the antibodies according to the inventionfor monotherapy or in combination with methotrexate or other DMARDs(Disease Modifying Anti-Rheumatic Drugs) for the treatment of adultswith moderate to severe active rheumatoid arthritis. It will beadministered as subcutaneous injection every 2 or 4 weeks. It will bechronic therapy in patients who have failed one or more DMARDs.Endpoints will include reduction in signs and symptoms and theinhibition of progression of structural damage in adult patients withactive rheumatoid arthritis. Prevention of disability, improvement insigns and symptoms measured by ACR criteria (ACR20>60%, ACR50>35%,ACR70>15%; index from the American College of Rheumatology;www.rheumatology.com).

A further embodiment of the invention is the use of the antibodiesaccording to the invention for the manufacture of medicaments for thetreatment of these diseases.

The invention relates also to the use of the antibodies as defined abovefor the manufacture of a pharmaceutical composition and comprises apharmaceutical composition containing an antibody according to theinvention with a pharmaceutically effective amount, optionally togetherwith a buffer and/or an adjuvant useful for the formulation ofantibodies for pharmaceutical purposes.

The invention further provides pharmaceutical compositions comprisingsuch antibodies in a pharmaceutically acceptable carrier. In oneembodiment, the pharmaceutical composition may be included in an articleof manufacture or kit.

The antibodies according to the invention are preferably produced byrecombinant means. Such methods are widely known in the state of the artand comprise protein expression in prokaryotic and eukaryotic cells withsubsequent isolation of the antibody polypeptide and usuallypurification to a pharmaceutically acceptable purity. For the proteinexpression, nucleic acids encoding light and heavy chains or fragmentsthereof are inserted into expression vectors by standard methods.Expression is performed in appropriate prokaryotic or eukaryotic hostcells like CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells,yeast, or E. coli cells, and the antibody is recovered from the cells(supernatant or cells after lysis).

Recombinant production of antibodies is well-known in the state of theart and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-61; Werner, R. G., et al., Arzneimittelforschung 48 (1998)870-80.

The antibodies may be present in whole cells, in a cell lysate, or in apartially purified or substantially pure form. Purification is performedin order to eliminate other cellular components or other contaminants,e.g. other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, column chromatography and others wellknown in the art. See Ausubel, F., et al., ed., Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987).

Expression in NSO cells is described by, e.g., Barnes, L.M., et al.,Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

The monoclonal antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of a human OX40L antibody areprepared by introducing appropriate nucleotide changes into the antibodyDNA, or by nucleotide synthesis. Such modifications can be performed,however, only in a very limited range, e.g. as described above. Forexample, the modifications do not alter the abovementioned antibodycharacteristics such as the IgG isotype and epitope binding, but mayimprove the yield of the recombinant production, protein stability orfacilitate the purification.

Any cysteine residue not involved in maintaining the proper conformationof the anti-OX40L antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cysteine bond(s) may be added to theantibody to improve its stability (particularly where the antibody is anantibody fragment such as an Fv fragment).

Nucleic acid molecules encoding amino acid sequence variants ofanti-OX40L antibodies are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of humanized anti-OX40Lantibody.

The invention also pertains to immunoconjugates comprising the antibodyaccording to the invention conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), aradioactive isotope (i.e., a radioconjugate). Conjugates of the antibodyand cytotoxic agent are made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters;(such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediatnine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitro-benzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta, E.S., et al., Science 238 (1987) 1098-1104). Carbon-14-labeled1-isothio-cyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO 94/11026.

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

In yet another aspect, the invention provides isolated B-cells from atransgenic non-human animal, e.g. a transgenic mouse, which express thehuman anti-OX40L antibodies (e.g. the parent antibodies produced by acell line selected from the group consisting of hybridoma cellsproducing antibodies according to the invention. Preferably, theisolated B cells are obtained from a transgenic non-human animal, e.g.,a transgenic mouse, which has been immunized with a purified orrecombinant form of OX40L antigen and/or cells expressing OX40L.Preferably, the transgenic non-human animal, e.g. a transgenic mouse,has a genome comprising a human heavy chain transgene and a human lightchain transgene encoding all or a portion of an antibody of theinvention. The isolated B-cells are then immortalized to provide asource (e.g. a hybridoma) of human anti-OX40L antibodies. Accordingly,the present invention also provides a hybridoma capable of producinghuman monoclonal antibodies according to the invention. In oneembodiment, the hybridoma includes a B cell obtained from a transgenicnon-human animal, e.g., a transgenic mouse having a genome comprising ahuman heavy chain transgene and a human light chain transgene encodingall or a portion of an antibody of the invention, fused to animmortalized cell.

In a particular embodiment, the transgenic non-human animal is atransgenic mouse having a genome comprising a human heavy chaintransgene and a human light chain transgene encoding all or a portion ofan antibody of the invention. The transgenic non-human animal can beimmunized with a purified or enriched preparation of OX40L antigenand/or cells expressing OX40L. Preferably, the transgenic non-humananimal, e.g. the transgenic mouse, is capable of producing isotypes ofhuman monoclonal antibodies to OX40L.

The human monoclonal antibodies according to the invention can beproduced by immunizing a transgenic non-human animal, e.g. a transgenicmouse, having a genome comprising a human heavy chain transgene and ahuman light chain transgene encoding all or a portion of an antibody ofthe invention, with a purified or enriched preparation of OX40L antigenand/or cells expressing OX40L. B cells (e.g. splenic B cells) of theanimal are then obtained and fused with myeloma cells to form immortal,hybridoma cells that secrete human monoclonal antibodies against OX40L.

In a preferred embodiment, human monoclonal antibodies directed againstOX40L can be generated using transgenic mice carrying parts of the humanimmune system rather than the mouse system. These transgenic mice,referred to herein as “HuMAb” mice, contain a human immunoglobulin geneminiloci that encodes unrearranged human immunoglobulin genes whichinclude the heavy (μ and γ) and κ light chain (constant region genes),together with targeted mutations that inactivate the endogenous μ and κchain loci (Lonberg, N., et al., Nature 368 (1994) 856-859).Accordingly, the mice exhibit reduced expression of mouse IgM or K, andin response to immunization, the introduced human heavy and light chaintransgenes undergo class switching and somatic mutation to generate highaffinity human IgG monoclonal antibodies (Lonberg, N., et al., Nature368 (1994) 856-859; reviewed in Lonberg, N., Handbook of ExperimentalPharmacology 113 (1994) 49-101; Lonberg, N., and Huszar, D., Intern.Rev. Immunol. 25 (1995) 65-93; and Harding, F., and Lonberg, N., Ann. N.Acad. Sci. 764 (1995) 536-546). The preparation of HuMAb mice isdescribed in Taylor, L., et al., Nucleic Acids Res. 20 (1992) 6287-6295;Chen, J., et al., Int. Immunol. 5 (1993) 647-656; Tuaillon, N., et al.,Proc. Natl. Acad. Sci. USA 90 (1993) 3720-3724; Choi, T. K., et al.,Nat. Genet. 4 (1993) 117-123; Chen, J., et al., EMBO J. 12 (1993)821-830; Tuaillon, N., et al., J. Immunol. 152 (1994) 2912-2920;Lonberg, N., et al., Nature 368 (1994) 856-859; Lonberg, N., Handbook ofExperimental Pharmacology 113 (1994) 49-101; Taylor, L., et al., Int.Immunol. 6 (1994) 579-591; Lonberg, N., and Huszar, D., Intern. Rev.Immunol. 25 (1995) 65-93; Harding, F., and Lonberg, N., Ann. N. Acad.Sci. 764 (1995) 536-546; Fishwild, D. M., et al., Nat. Biotechnol. 14(1996) 845-851, the contents of all of which are hereby incorporated byreference in their entirety. See further, U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877, 397; 5,661,016;5,814,318; 5,874,299; 5,545,807; 5,770,429; WO 98/24884; WO 94/25585; WO93/1227; WO 92/22645; and WO 92/03918.

To generate fully human monoclonal antibodies to OX40L, HuMAb mice canbe immunized with a purified or enriched preparation of OX40L antigenand/or cells expressing OX40L in accordance with the general method, asdescribed by Lonberg, N., et al., Nature 368 (1994) 856-859; Fishwild,D. M., et al., Nat. Biotechnol. 14 (1996) 845-851 and WO 98/24884.Preferably, the mice will be 6-16 weeks of age upon the firstimmunization. For example, a purified or enriched preparation of solubleOX40L antigen (e.g. purified from OX40L-expressing cells) coupled to KLHor in PBS can be used to immunize the HuMAb mice intra-peritoneally.This can be combined by alternate immunization with the isolated OX40Lprotein with cells expressing OX40L, e.g., a tumor cell line, to promoteimmune responses. Cumulative experience with various antigens has shownthat the HuMAb transgenic mice respond best when initially immunizedintra-peritoneally (i.p.) with antigen in complete Freund's adjuvant,followed by every other week i.p. immunizations (for example, up to atotal of 6) with antigen in incomplete Freund's adjuvant. The immuneresponse can be monitored over the course of the immunization protocolwith plasma samples being obtained by retroorbital bleeds. The plasmacan be screened by ELISA, and mice with sufficient titers of anti-OX40Lhuman immunoglobulin can be used for immortalization of corresponding Bcells. Mice can be boosted intravenously with antigen 3 to 4 days beforesacrifice and removal of the spleen and lymph nodes. Several mice willbe immunized for each antigen. For example, a total of twelve HuMAb miceof the HCo7 and HCo12 strains can be immunized.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen, J., et al., EMBO J. 12 (1993)821-830), a CMD disruption in their endogenous heavy chain genes (asdescribed in Example 1 of WO 01/14424), a KCo5 human kappa light chaintransgene (as described in Fishwild, D. M., et al., Nat. Biotechnol. 14(1996) 845-851), and a HCo7 human heavy chain transgene (as described inU.S. Pat. No. 5,770,429).

The HCo12 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen, J., et al., EMBO J. 12 (1993)821-830), a CMD disruption in their endogenous heavy chain genes (asdescribed in Example 1 of WO 01/14424), a KCo5 human kappa light chaintransgene (as described in Fishwild, D. M., et al., Nat. Biotechnol. 14(1996) 845-851), and a HCo12 human heavy chain transgene (as describedin Example 2 of WO 01/14424).The mouse lymphocytes can be isolated andfused with a mouse myeloma cell line using PEG based on standardprotocols to generate hybridomas. The resulting hybridomas are thenscreened for the production of antigen-specific antibodies. For example,single cell suspensions of splenic and lymph node-derived lymphocytesfrom immunized mice are fused to one-sixth the number of SP 2/0nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG. Cellsare plated at approximately 2×10⁵ in flat bottom microliter plate,followed by about two weeks incubation in selective medium.

Individual wells are then screened by ELISA for human anti-OX40Lmonoclonal IgM and IgG antibodies. Once extensive hybridoma growthoccurs, medium is analyzed, usually after 10-14 days. The antibodysecreting hybridomas are replated, screened again, and if still positivefor human IgG, anti-OX40L monoclonal antibodies, can be subcloned atleast twice by limiting dilution. The stable subclones are then culturedin vitro to produce antibody in tissue culture medium forcharacterization.

The assay tree principally is composed of an unspecific assay on IgG(“IgG-ELISA”) followed by a specific ELISA and apparent FACS assay fordetermination of antigen binding to either purified OX40L protein orOX40L-expressing cells. The next step comprises functional assays wherethe competition of the anti OX40L antibody with its natural interactionpartner e.g. soluble, purified OX40 for either purified OX40L or OX40Lexpressed on cells is determined, e.g. competition ELISA or FACS. Thenext step comprises a functional assay where the blocking capability ofanti-OX40L antibody of OX40-signal transduction is determined, e.g.NFκB-activation “NFκB-assay”). The next step comprises functional assayswhere the blocking capability of the anti-OX40L antibody concerningT-cell activation is determined (“T-cell activation assay”and“TT-assay”).

Because CDR sequences are responsible for antibody-antigen interactions,it is possible to express recombinant antibodies according to theinvention by constructing expression vectors that include the CDRsequences according to the invention onto framework sequences from adifferent human antibody (see, e.g., Riechmann, L., et al., Nature 332(1998) 323-327; Jones, P., et al., Nature 321 (1986) 522-525; and Queen,C., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline human antibody gene sequences. These germline sequenceswill differ from mature antibody gene sequences because they will notinclude completely assembled variable genes, which are formed by V(D)Jjoining during B cell maturation. Germline gene sequences will alsodiffer from the sequences of a high affinity secondary repertoireantibody at individual evenly across the variable region.

The invention further comprises the use of an antibody according to theinvention for the diagnosis of OX40L in vitro, preferably by animmunological assay determining the binding between OX40L (eithersoluble or membrane-bound) of a sample and the antibody according to theinvention.

In another aspect, the present invention provides a composition, e.g. apharmaceutical composition, containing one or a combination of humanmonoclonal antibodies, or the antigen-binding portion thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. More specifically, the composition is apharmaceutical or a diagnostic composition and even more specificallythe pharmaceutical composition comprises an antibody as defined aboveand at least one pharmaceutically acceptable excipient. The compositionmust be sterile and fluid to the extent that the composition isdeliverable by syringe.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion). Preferablysuch a carrier is an aqueous pH buffered solution (for example acetate,citrate, phosphate or histidine), preferably isotonic, preferablycontaining in addition an inorganic salt, sugar, polyol and/or asurfactant. Pharmaceutical acceptable carriers are also such asdescribed in Remington's Pharmaceutical Sciences, 16^(th) edition, Osol,A. Ed. (1980).

The antibody concentration is preferably from 0.1 mg/ml to 50 mg/ml.Preferably the pH value of the buffered solution ranges from 4.0 to 8.0at a buffer concentration of 1 mM to 200 mM. Preferred salts are sodiumchloride and/or sodium phosphate in the range of 1 mM to 200 mM.Preferred sugars are sucrose and/or trehalose in the range of 1% to 15%(w/v). Preferred polyols are glycerol, propylene glycol, liquidpolyethylene glycol, and/or the like in the range of 1% to 15% (w/v).The surfactant is preferably a polysorbate (e.g. polysorbate 20 or 80)and/or poloxamere in the range of 0.001% to 0.5% (w/v). A preferredpharmaceutical composition contains antibody from 0.1 mg/ml to 50 mg/mland 1 mM to 200 mM phosphate buffered saline pH 4.0 to 8.0.

A composition of the present invention can be administered by a varietyof methods known in the art to a patient with the need thereof. As willbe appreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results.

Pharmaceutically acceptable excipients or carriers include sterileaqueous solutions or sterile powders for the extemporaneous preparationof sterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A typical weekly dosagemight range from about 0.1 mg/kg to about 20 mg/kg or more, depending onthe factors mentioned above.

The following examples, references, sequence listing and figures areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention. Abbreviations:

Amino acids are abbreviated either in the three (Leu) or one letter code(L). S228P means an exchange of Serin to Proline at position 228 of IgG4heavy chain. L234 means amino acid leucine at position 234 according toEU numbering (Kabat). L234A means amino acid leucine at position 234 ischanged to alanine. L235A means amino acid leucine at position 235 ischanged to alanine. PVA236 means that in the 236 region ELLG of IgG1 orEFLG of IgG4 is amended in PVA. GLPSS331 means that in the 331 regionALPAP of IgG1 or GLPAP of IgG2 is changed to GLPSS. Delta G236 meansamino acid at position 236 is deleted. IgG4× means mutation S228P inIgG4. LC2010-001 is a synonym for LC.001 Fcg is synonymous for Fcgamma(Fcγ)

Other sequence amendments of antibodies are designated analogously.

Recombinant soluble human OX40L hOX40L-His fused to a Histidine tagRecombinant soluble murine OX40L mOX40L-His fused to a Histidine tagRecombinant soluble human OX40L hOX40L-Flag fused to a Flag tagRecombinant soluble murine OX40L mOX40L-Flag fused to a Flag tagRecombinant soluble human OX40 hOX40-hFc fused to human Fcγ Rabbitanti-murine Fcγ monoclonal Anti-mFc antibody Goat anti-human Fcγmonoclonal Anti-hFc antibody Murine anti-histidine monoclonal Anti-Hisantibody Recombinant soluble human OX40 hOX40-mFc fused to murine FcγMurine anti-TNFα monoclonal Anti-TNFa antibody Murine anti-CD40Lmonoclonal Anti-CD40L antibody Tumor Necrosis Factor alpha TNFα CD40Ligand CD40L Rat anti-human OX40L monoclonal TAG34 antibody Humananti-human OX40L LC.001 monoclonal antibody Human anti-human OX40LLC.005 monoclonal antibody Human anti-human OX40L LC.010 monoclonalantibody Human anti-human OX40L LC.019 monoclonal antibody Humananti-human OX40L LC.029 monoclonal antibody Human anti-human OX40LLC.033 monoclonal antibody phytohemagglutinin PHA

EXAMPLES Example 1 Generation of a Hybridoma Cell Line ProducingAnti-OX40L Antibodies

Culture of hybridomas: HuMab hybridomas were cultured in IMDM (Cambrex),Fetal clone 1 Bovine serum (Perbio Science), origin Hybridoma cloningfactor (Igen), sodium pyruvate, penicillin/streptomycin,2-mercaptoethanol, HAT (Sigma-Aldrich) and Kanamycin (Invitrogen) in 37°C. and 5% CO₂.

Immunization procedure of transgenic mice: LC2010-001: Six HCo7 (2 malesand 4 females), strain GG2201 (Medarex, San José, Calif., USA), and 4HCo12 (4 males), strain GG2198 (Medarex, San José, Calif., USA) werealternatingly immunized with 1×10⁶ HEK293 cells, transiently transfectedwith an expression vector for human OX40L (hOX40L), and 20 μg solubleextracellular domain of hOX40L. Eight immunizations were performed intotal, four intraperitoneal (i p) immunizations with the hOX40Lexpressing cells and four subcutaneous (s.c.) immunizations at the tailbase with recombinant protein. For the first immunization, 100 μl of1×10⁶ HEK293-hOX40L cells was mixed with 100 μl complete Freund'sadjuvant (CFA; Difco Laboratories, Detroit, USA). For all otherimmunizations, 100 μl of cells in PBS were used or recombinant proteinwas mixed with 100 μl incomplete Freund's adjuvant (ICFA; Difco).

When serum titers of anti-hOX40L were found to be sufficient, mice wereadditionally boosted twice with 15 μg hOX40L extracellular domain in 200μl PBS intravenously (i.v.) 4 and 3 days before fusion. LC2010-001 wasderived from one of the HCo12 mice.

LC2010-005, -010, -019, -029 and -033: Five HCo7 (4 males and 1 female),strain GG2201 (Medarex, San José, Calif., USA) were immunized with 20 μgsoluble extracellular domain of hOX40L. Seven immunizations wereperformed in total, four intraperitoneal (i.p.) and three subcutaneous(s.c.) immunizations at the tail base. For the first immunization, 100μl of recombinant protein was mixed with 100 μl complete Freunds'adjuvant (CFA; Difco Laboratories, Detroit, USA). For all otherimmunizations, 100 μl of recombinant protein was mixed with 100 μlincomplete Freunds' adjuvant (ICFA; Difco).

When serum titers of anti-hOX40L were found to be sufficient, mice wereadditionally boosted twice with 15 μg hOX40L extracellular domain in 200μl PBS intravenously (i.v.) 4 and 3 days before fusion.

Hybridoma generation: Mice were sacrificed and the spleen and lymphnodes flanking the abdominal aorta and vena cava were collected. Fusionof splenocytes and lymph node cells with the fusion partner SP 2.0 cellswas performed according to standard operating procedures.

Antigen specific ELISA: Anti-OX40L titers in sera of immunized mice weredetermined by antigen specific ELISA. Plate (96 flat bottom ELISA plate,Greiner) was coated with 0.1 μg/ml purified OX40L dissolved in PBS andcoated overnight at room temperature. Thereafter, wells were blockedwith PBSTC (PBS containing 0.05% Tween 20 (Sigma-Aldrich Chemie BV) and2% chicken serum (Gibco)) for 1 hour at room temperature.

Tested serum taps were diluted 1:50 in PBSTC and added to the wells.Serum obtained from mice prior to immunization was dissolved 1:100 inPBSTC and used as negative control. A mouse antibody directed againsthuman OX40L was dissolved 1:50 in PBSTC and used as a positive control.Plates were incubated for 1 hour at room temperature. Subsequently,plates were washed twice using PBST (PBS containing 0.05% Tween 20.Gt-α-huIgG-HRP (Jackson) was diluted 1:5000 in PBSTC and added to thewells containing the tested taps and the negative control. Rb-α-mIgG(Jackson) was diluted 1:3000 in PBSTC and added to the wells containingthe positive control. Plates were incubated for 1 hour at roomtemperature. Finally, plates were washed three times using PBST anddeveloped with freshly prepared ABTS® solution (1 mg/ml) (ABTS:2,2′-azino bis (3-ethyl-benzthiazoline-6-sulfonic acid) for 30 minutesat room temperature (RT) in the dark. Absorbance was measured at 405 nm.

kappa-ELISA: To determine whether hybridomas that resulted from thefusion generate human antibodies, a kappa-ELISA was performed. ELISAplates were coated with rat anti-human IgG kappa-light chain antibody(DAKO) diluted 1/10000 in PBS by overnight incubation at 4° C. Afterdiscarding the wells, plates were blocked by incubation with PBSTC(PBSC, supplemented with 0.05% Tween-20 (PBSTC)) for 1 hour at roomtemperature. Thereafter, wells were incubated with hybridoma culturesupernatant, ½ diluted in PBSTC. Culture medium ½ diluted in PBSTC wasused as negative control, kappa-light positive mouse serum 1/100 dilutedin PBSTC served as positive control. Subsequently, wells were washedthrice and were incubated with HRP-conjugated rat anti-human IgG F(ab′)₂(DAKO), diluted 1/2000 in PBSTC for 1 h at 37° C. Wells were washedthrice and assays were developed with freshly prepared ABTS® solution (1mg/ml) for 30 minutes at room temperature (RT) in the dark. Absorbancewas measured at 405 nm in an ELISA plate reader.

Example 2 Cloning and Sequence Analysis of Anti-OX40L HuMab VariableDomains

kappa-light and γ1-heavy chains: The nucleotide sequences coding for thelight chain variable region VL and the heavy chain variable region VH ofthe OX40L HuMabs were isolated by a standard cDNA synthesis/PCRprocedure.

Total RNA was prepared from 1×10⁶-1×10⁷ hybridoma cells using theGeneRacer™ Kit (Invitrogen). Hybridoma derived RNA was used as atemplate for the 1^(st) strand cDNA synthesis and ligation of theGeneRacer™ Oligo-dT Primer. 2^(nd)-strand cDNA synthesis and further PCRamplification of V_(L) and V_(H) encoding cDNA fragments were performedwith reverse light and heavy chain primers complementary to nucleotidesequences of the kappa-light and γ1-heavy chain constant region and5′-specific GeneRacer™ primers, respectively. The PCR products werecloned using the TOPO™ TA cloning kit from Invitrogen™ Life Technologiesand pCR4-TOPO™ as a cloning vector. Cloned PCR products were identifiedby restriction mapping of the appropriate plasmids using EcoRI fordigestion and expected/calculated DNA fragment sizes of about 740 and790 by for V_(L) and V_(H), respectively.

The DNA sequence of cloned PCR fragments was determined by double strandsequencing.

The GCG (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Vector-NTI 8 (InforMax, Inc) was used for general dataprocessing. DNA and protein sequences were aligned using the GCG moduleCLUSTALW. Sequence alignments were made using the program GENEDOC(version 2.1).

Example 3 Construction of Expression Plasmids for an Anti-OX40L IgG1HuMab

The anti-OX40L HuMab light and heavy chain encoding genes wereseparately assembled in mammalian cell expression vectors.

Thereby the gene segments encoding the anti-OX40L HuMab light chainvariable region (V_(L)) and the human kappa-light chain constant region(C_(L), SEQ ID NO:13) were joined as were gene segments for theanti-OX40L HuMab heavy chain variable region (V_(H)) and the humanγ1-heavy chain constant region (C_(H1)-Hinge-C_(H2)-C_(H3), SEQ IDNO:14).

General information regarding the nucleotide sequences of human lightand heavy chains from which the codon usage can be deduced is given in:Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,fifth ed., NIH Publication No. 91-3242 (1991).

The transcription unit of the anti-OX40L HuMab kappa-light chain iscomposed of the following elements:

-   -   The immediate early enhancer and promoter from the human        cytomegalovirus (HCMV),    -   A synthetic 5′-UT including a Kozak sequence,    -   A murine immunoglobulin heavy chain signal sequence including        the signal sequence intron,    -   The cloned anti-OX40L HuMab variable light chain cDNA arranged        with a unique BsmI restriction site at the 5′ end and a splice        donor site and a unique NotI restriction site at the 3′ end,    -   The genomic human kappa-gene constant region, including the        intron 2 mouse Ig-kappa enhancer (Picard, D., and Schaffner, W.,        Nature 307 (1984) 80-82) and    -   The human immunoglobulin kappa-polyadenylation (“poly A”) signal        sequence.

The transcription unit of the anti-OX40L HuMab γ1-heavy chain iscomposed of the following elements:

-   -   The immediate early enhancer and promoter from the human        cytomegalovirus (HCMV),    -   A synthetic 5′-UT including a Kozak sequence,    -   A modified murine immunoglobulin heavy chain signal sequence        including the signal sequence intron,    -   The cloned anti-OX40L HuMab variable heavy chain cDNA arranged        with a unique BsmI restriction site at the 5′ and a splice donor        site and a unique NotI restriction site at the 3′ end,    -   The genomic human γ1-heavy gene constant region, including the        mouse Ig μ-enhancer (Neuberger, M. S., EMBO J. 2 (1983)        1373-1378),    -   The human γ1-immunoglobulin polyadenylation (“poly A”) signal        sequence.

Functional elements of the anti-OX40L HuMab kappa-light chain andγ1-heavy chain expression plasmids: Beside the anti-OX40L HuMabkappa-light chain or γ1-heavy chain expression cassette these plasmidscontain

-   -   A hygromycin resistance gene    -   An origin of replication, oriP, of Epstein-Barr virus (EBV)    -   An origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   A β-lactamase gene which confers ampicillin resistance in E.        coli.

Example 4 Construction of Expression Plasmids for an Anti-OX40L IgG4HuMab

An anti-OX40L γ1-heavy chain prototype expression plasmid was derivedfrom the anti-OX40L γ1-heavy chain expression plasmid by replacing thehuman genomic γ1-constant region and γ1-immunoglobulin polyadenylation(“poly A”) signal sequence by the human genomic γ4-constant region (SEQID NO:15) and γ4-immunoglobulin polyadenylation-signal sequence.

For the expression of anti-OX40L HuMab kappa-light chains the sameexpression plasmids were used as described for IgG1 (see above).

Example 5 Construction of Expression Plasmids for Mutant (variant)anti-OX40L IgG1 and IgG4 based on LC.001

Expression plasmids encoding mutant anti-OX40L γ1- and γ4-heavy chainswere created by site-directed mutagenesis of the wild type expressionplasmids using the QuickChange™ Site-Directed mutagenesis Kit(Stratagene). Amino acids are numbered according to EU numbering(Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85;Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,5^(th) ed., Public Health Service, NIH Publication No. 91-3242,Bethesda, Md. (1991)).

TABLE 1 Abbre- Isotype viation Mutations Description IgG1 IgG1v1PVA-236; The amino acid sequence GLPSS331 Glu₂₃₃Leu₂₃₄Leu₂₃₅Gly₂₃₆ ofthe human as specified γ1-heavy chain is replaced by the amino by acidsequence Pro₂₃₃Val₂₃₄Ala₂₃₅ of the E233P; human γ2-heavy chain. L234V;The amino acid sequence L235A; Ala₃₂₇Leu₃₂₈Pro₃₂₉Ala_(330P)Pro₃₃₁ of thedelta G236; human γ1-heavy chain is replaced by the A327G; amino acidsequence A330S; Gly₃₂₇Leu₃₂₈Pro₃₂₉Ser₃₃₀Ser₃₃₁ of the P331S humanγ4-heavy chain. IgG1 IgG1v2 L234A; The amino acid sequence Leu₂₃₄Leu₂₃₅L235A of the human γ1-heavy chain is replaced by the amino acid sequenceAla₂₃₄Ala₂₃₅ IgG4 IgG4v1 S228P; Ser₂₂₈ of the human γ4-heavy chain isL235E replaced by Pro₂₂₈ and Leu₂₃₅ of the human γ4-heavy chain isreplaced by Glu₂₃₅ IgG4 IgG4x S228P Ser₂₂₈ of the human γ4-heavy chainis replaced by Pro₂₂₈

Example 6 Production of Recombinant Anti OX40L HuMabs

Recombinant HuMabs were generated by transient transfection of adherentHEK293-EBNA cells (ATTC CRL-10852) cultivated in DMEM (Gibco)supplemented with 10% ultra-low IgG FCS (Gibco), 2 mM Glutamine (Gibco),1% v/v nonessential aminoacids (Gibco) and 250 μg/ml G418 (Roche). Fortransfection Fugene™ 6 (Roche) Transfection Reagent was used in a ratioof reagent (μl) to DNA (μg) ranging from 3:1 to 6:1. Immunoglobulinlight and heavy chains were expressed from two different plasmids usinga molar ratio of light chain to heavy chain encoding plasmid from 1:2 to2:1. HuMab containing cell culture supernatants were harvested at day 4to 11 after transfection. Supernatants were stored at −20° C. untilpurification.

General information regarding the recombinant expression of humanantibody in e.g. HEK293 is given in: Meissner, P., et al., Biotechnol.Bioeng. 75 (2001) 197-203.

Example 7 Affinity Analysis of Antibodies TAG34, LC.001, LC.005, LC.010,LC.019, LC.029, LC.033

Instrument: Biacore 3000, running and reaction buffer: HBS-P (10 mMHEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25° C. Injection ofanalyte was performed at 7 concentrations between 0.78 nM and 100 nM for3 minutes and washed with HBS-P for 5 minutes. Regeneration of thesurface (carboxymethylated dextrane surface, CM) was performed by twoinjections of 10 mM Glycine pH 2.0 for 1 min each. The chip, assayformat and sequence of injections and kinetic data correspond to thedescription in the following table. Kinetic data were calculated byfitting kinetic data to a 1:1 Langmuir binding model.

TABLE 2 Chip Capturing Ligand Analyte ka (1/Ms) kd (1/s) K_(D) (M) CM5Anti-mFcg TAG34 hOX40L-His 8.84 × 10⁴ 3.32 × 10⁻⁵ 3.75 × 10⁻¹⁰ CM5Anti-hFcg LC.001 hOX40L-His 9.01 × 10⁴ 7.16 × 10⁻⁹ <1.1 × 10⁻¹¹ CM5Anti-hFcg LC.005 hOX40L-His 6.84 × 10⁴ 2.02 × 10⁻⁷ <1.5 × 10⁻¹¹ CM5Anti-hFcg LC.010 hOX40L-His 6.25 × 10⁴  2.5 × 10⁻⁵ 3.99 × 10⁻¹⁰ CM5Anti-hFcg LC.019 hOX40L-His 7.89 × 10⁴ 7.53 × 10⁻⁸ <1.2 × 10⁻¹¹ CM5Anti-hFcg LC.029 hOX40L-His 1.41 × 10⁵  2.4 × 10⁻⁸ <7.1 × 10⁻¹² CM5Anti-hFcg LC.033 hOX40L-His 7.01 × 10⁴ 2.09 × 10⁻⁷ <1.4 × 10⁻¹¹

No interaction between TAG34 and mOX40L could be measured.

Data Evaluation and Data Deposition for all Biacore assays: Negativecontrol data (e.g. buffer curves) were subtracted from sample curves forcorrection of system intrinsic baseline drift and for noise signalreduction. BiaEvaluation version 4.01 was used for analysis ofsensorgrams and for calculation of affinity data.

Example 8 Inhibitory Competition Assay of Anti-hOX40L AntibodiesInhibiting the Interaction of hOX40L With Immobilized hOX40

Instrument: Biacore 3000, running and reaction buffer: HBS-P (10 mMHEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25° C. Prior to injectionthe analyte (10 nM) and competitor (eight concentrations between 0.78 nMand 100 nM) were preincubated for at least 20 min at 22° C. Injection ofanalyte +/− competitor was performed for 3 minutes and washed with HBS-Pfor three minutes. Regeneration of the surface was performed by twoinjections of 10 mM Glycine pH 2.0 for 1 min each. The chip, assayformat and sequence of injections and kinetic data correspond to thedescription in the following table 3.

TABLE 3 Chip Ligand Analyte Competitor IC50 (M) CM5 OX40-hFc hOX40L-HisTAG34 7 × 10⁻⁹ CM5 OX40-hFc hOX40L-His LC.001 4 × 10⁻⁹ CM5 OX40-hFchOX40L-His LC.005 3 × 10⁻⁹

All antibodies inhibit the binding of OX40L to OX40 in solution(solution affinity). LC.001 and LC.005 show a lower IC50-value thanTAG34.

Example 9 Epitope Characterization of Anti-OX40L Antibodies TAG34,LC.001, LC.005, LC.010, LC.019, LC.029, LC.033

Instrument: Biacore 3000, running and reaction buffer: HBS-P (10 mMHEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25° C. The epitope groupswere determined by cross competition between the listed antibodies.Prior to injection the analyte (50 nM) and competitor (100 nM) werepreincubated for at least 20 min at 22° C. Injection of analyte +/−competitor for two minutes, wash with HBS-P for three minutes.Regeneration of the surface was performed by two injections of 10 mMGlycine pH 2.0 for 1 min each. The chip, assay format and sequence ofinjections and kinetic data correspond to the description in thefollowing table 4.

TABLE 4 Chip Capturing Ligand Analyte Competitor Epitope CM5 Anti-hFcgAnti-OX40L hOX40L-His TAG34 A (A, B, C) CM5 Anti-hFcg Anti-OX40LhOX40L-His LC.001 A (A, B, C) CM5 Anti-hFcg Anti-OX40L hOX40L-His LC.005B (A, B, C) CM5 Anti-hFcg Anti-OX40L hOX40L-His LC.010 B (A, B, C) CM5Anti-hFcg Anti-OX40L hOX40L-His LC.019 A/B (A, B, C) CM5 Anti-hFcgAnti-OX40L hOX40L-His LC.029 B (A, B, C) CM5 Anti-hFCg Anti-OX40LhOX40L-His LC.033 A (A, B, C)

The OX40L epitope recognized by TAG34 was defined as epitope A.Antibodies within one epitope group (A or B) show cross inhibitoryactivity, while antibodies from different groups show additive bindingsignals. LC.019 neutralizes other antibodies from group A as well asfrom group B.

Example 10 Binding Specificity of TAG34, LC.001 and LC.005 to CD40L andTNFα

Instrument: Biacore 3000, running and reaction buffer: HBS-P (10 mMHEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25° C. Injection ofanalyte was performed for three minutes at 100 nM and 500 nM and washedwith HBS-P for two minutes. Regeneration of the surface was performed bytwo injections of 100 mM HCl for 1 min each. The chip, assay format andsequence of injections and kinetic data correspond to the description inthe following table 5.

TABLE 5 Chip Capturing Ligand Analyte CM5 Anti-mFcg TAG34 TNFα Anti-hFcgLC.001 CD40L LC.005 OX40L Anti-TNFα Anti-CD40L

In this assays CD40L showed some unspecific binding to all antibodies orto the chip surface, but after subtraction of background signals thisassay showed, that there was no unspecific binding of TNFα and CD40L (upto 500 nM) to the immobilized antibodies TAG34, LC.001 and LC.005.

Example 11 Affinity Analysis of Antibodies LC.001-IgG1 and LC.001-IgG4×

Instrument: Biacore 3000, running and reaction buffer: HBS-P (10 mMHEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25° C. Injection ofanalyte was performed at eight concentrations of 0.78 nM-100 nM forthree minutes and washed with HBS-P for five minutes. Regeneration ofthe surface was performed by two injections of 100 mM HCl for 1 mM each.The chip, assay format and sequence of injections and kinetic datacorrespond to the description in the following table. Kinetic data werecalculated by fitting kinetic data to a 1:1 Langmuir binding model.

TABLE 6 Chip Capturing Ligand Analyte ka (1/Ms) kd (1/s) K_(D) (M) CM5Anti-mFcg LC.001 hOX40L- 4.27 × 10⁴ 3.46 × 10⁻⁸  <2.3 × 10⁻¹¹ His CM5Anti-mFcg LC.001- hOX40L- 4.85 × 10⁴ 7.72 × 10⁻⁸ <2.06 × 10⁻¹¹ IgG4x His

LC.001 and LC.001-IgG4× show the same affinity to hOX40L-His.

Example 12 ELISA Assay for Detection of Antibodies Binding to OX40L

SA-coated plates (96 flat bottom ELISA plate, Microcoat) were coatedwith 0.5 μg/ml biotinylated OX40L dissolved in incubation buffer (IB=PBScontaining 0.1% Tween 20 (Serva) and 1% blocking protein) for 1 hour atroom temperature. Then the plates were washed twice using washing buffer(WB=saline containing 0.1% Tween 20).

Samples (cell culture supernatants or purified antibodies) were seriallydiluted in IB and added to the wells. Plates were incubated for 1 hourat room temperature. Subsequently, plates were washed twice using WB.Subsequently a conjugate of a goat antibody against human IgG and POD(Dianova) was diluted to 50 ng/ml in IB and added to the wells. Plateswere incubated for 1 hour at room temperature. Finally, plates werewashed twice using WB and developed with ready-to-use ABTS® solution(Roche) at room temperature (RT) in the dark. Absorbance was measured at405 nm after absorbance of the highest concentration reached asufficient OD (FIG. 1 a). EC50 values are obtained in the range of 3nM-8 nM.

Example 13

ELISA assay for Detection of Antibodies Inhibiting Interaction of HumanOX40/Human OX40L

SA-coated plates (96 flat bottom ELISA plate, Microcoat, Germany) werecoated with 0.5 μg/ml biotinylated OX40L dissolved in IB for 1 hour atroom temperature. Then the plates were washed twice using WB (PBSbuffer, 0.1% (w/v) Tween™ 20.

Samples were diluted in IB to a concentration of 1 μg/ml and added tothe wells in serial dilutions. In order to achieve maximum binding ofOX40 to OX40L in some well only IB was added. Then to each well asolution of human OX40 conjugated with Digoxigenin (Roche DiagnosticsGmbH, DE) at a concentration of 0.2 μg/ml was added. Plates wereincubated for 1 hour at room temperature. Subsequently, plates werewashed twice using WB. Sheep<Digoxigenin>-POD (Roche) was diluted to 50mU/ml in IB and added to the wells. Plates were incubated for 1 hour atroom temperature. Finally, plates were washed twice using WB anddeveloped with ready-to-use ABTS® solution (Roche) at room temperature(RT) in the dark. Absorbance was measured at 405 nm after 10 to 20minutes (FIG. 1 b). IC50 values were obtained in the range between 1 nMand 4 nM.

Example 14 FACS-Assay for Detection of HuMabs Inhibiting Interaction ofHuman OX40 With Human OX40L Expressed on K562 Cells (K562_OX40L Cells)

Purpose: Assay for determination of the HuMab hOX40L's property to blockinteraction of Dig-labeled hOX40:hFc fusion protein with hOX40Lexpressing cell line K562_hOX40L.

Procedure: The assay is performed with Dig labeled hOX40:hFc as “assayreagent” and the HuMab hOX40L as “competitor”.

Assay reagent: stock 0.5 μg/μl-1:10 diluted in PBS), 100 μl antidigoxigenin-FLUOS. 1:25 diluted in PBS/0.5% BSA/1% blocking reagent(Roche Diagnostics GmbH, DE).

2×10⁵ K562_OX40L cells (grown in ISF-0) were washed in 2 ml PBS andresuspended in 100 μl PBS. This is followed by the addition ofcompetitor in PBS (competitor/reagent relation of0:1/1:1/1.5:1/2:1/2.5:2/5:1). This is followed by an incubation time of30 Min, RT and day light. Then reagent (in PBS) was added; incubationtime: 30 Min, RT and day light. The cells were washed with 2 ml PBS andpelleted by centrifugation. The secondary antibody for staining(anti-digoxigenin-fluorescein, Fab-Fragments (Roche, 1207741)) was addedand incubated for 30 Min, 4° C. in the dark. The cells were washed with2 ml PBS and pelleted by centrifugation. After that the cells wereresuspended in 0.5 ml PBS. Measurement of the samples was performed in aFACS-Scan (FIG. 2).

Example 15 Functional Assay for Determination of the Inhibitory Capacityof Antibodies for hOX40/hOX40L Signaling (“NFκB-Assay”)

HeLa wild type (wt) and HeLa cells expressing human OX40 (HeLa_OX40)were grown in Minimal Essential Medium (MEM), 1× Na-pyruvate, 1× NonEssential Aminoacids (Gibco), 10% FCS and in the case of the recombinantcells+600 μg/ml G418. K562 and K562 expressing OX40L were grown in ISF-Omedium, and in the case of recombinant cells 200 μg/ml G418 was added.

HeLa wt or HeLa_OX40 cells were seeded at a cell density of 3×10⁴cells/100 μl in a 96-well plate w/o G418 and incubated over night in aCO₂-incubator. K562_wt or K562_OX40L cell were added in a cell-to-cellrelation of 1:1. Formalin-fixed or not formalin-fixed K562 cellsexpressing OX40L (frozen at −70° C.) were thawed and diluted 1:10 inMEM/10% FCS; the K562_OX40L cells were preincubated with antibodyagainst OX40L for 30 min at RT. The stimulation time with the K562_OX40Lcells was between 30 and 150 min. Protein extraction from cell nucleitook place according suppliers instruction with the NE-Kit from ActiveMotif. The TransAM NFκB-ELISA from Active Motif (the assay was performedaccording suppliers instructions) was used to determine OX40-signaling,which results in NFκB activation. The measurement was performed atwavelength 450/620 rim absorption in a Tecan MTP-Reader (FIG. 3). IC50values were obtained in the range between 1 and 5 nM.

Example 16 T-Cell Activation Assay

Assay principle: Human peripheral blood mononuclear cells (PBL) areactivated with a sub-optimal concentration of the T-cell mitogenphyto-hemagglutinin (PHA), and co-stimulated with K562 cellsoverexpressing OX40L. Under the assay conditions, activated T-cellsincubated 24 hr at 37° C. produce IL-2. The cytokine is measured in thesupernatant using an ELISA assay. To determine the blocking effect of aMab, the K562_OX40L cells are pre-incubated for 1 hour with appropriatedilutions of the antibody before co-culture with PBL.

Procedure: Human peripheral blood mononuclear cells (PBL) are separatedfrom heparinized whole blood by density gradient centrifugation inHistopaque®-1077 (Sigma). After washing with Hanks, cells are countedusing Turk's solution, and the cell are resuspended at a concentrationof 10⁶/ml in RPMI 1640 (Gibco), supplemented with penicillin,streptomycin and glutamine (Gibco 10378-016), and 10% FBS. K562 controlcells (wild type) are maintained in the same RPMI medium supplemented asdescribed. K562 cells transfected with OX40L are maintained in the samemedium supplemented with Geneticin (G418, Gibco) at a finalconcentration of 50 mg/ml. K562 cells (either WT or OX40L+) are dilutedwith the same medium at 1.5×10⁵ cells/ ml, and dispensed into each wellof a 96 well tissue culture plate at 50 μl/well (0.75×104/well).Appropriate dilutions of the Mab are added to the cells in a volume of20 μl/well, and incubated for 1 hr. at 37° C. Each dilution is tested induplicate wells. PBL are added at a volume of 100 μl/well (10⁵cell/well). The final ratio of PBL to K562 cells is ˜13:1. PHA (10×)(Sigma L-9132) is added at 20 μl/well (final concentration 0.75 μg/ml).The total volume per well is completed to 200 μl with RPMI/10% FCS.Plates are incubated at 37° C. in a 5% CO₂-humidified incubator for 24hrs. After centrifugation of the plates, the supernatants are collectedand IL-2 tested by ELISA (BD, San Diego, Calif., Cat No2627KI),according to the manufacturer' specifications (FIG. 4). To calculate theIC₅₀ (Mab concentration that blocks 50% of IL-2 release byOX40L-stimulated PBL), the background IL-2 concentration produced in thecontrol cultures (PBL+PHA+K562WT) was subtracted from the total IL-2produced by PBL-stimulated with K562xOX40L+ cells (FIG. 5). IC₅₀ TAG34:0.07 μM; LC.001: 2 nM; LC.005: 10 nM. The IC₅₀ values are in the rangebetween 2 and 10 nM.

Example 17 Tetanus assay (‘TT-Assay’) Testing the Inhibitory Effect ofAntibodies on Peripheral Blood Lymphocytes Stimulated by Tetanus Toxoid

Peripheral blood mononuclear cells (PBMC) were isolated from heparinizedblood by Ficoll Hypaque. In most cases freshly isolated PBMC were usedfor this assay. In some cases also cryopreserved PBMC were used. Themedium for this assay was RPMI containing 10% human male AB serum(Sigma-Aldrich); 2 mM Glutamine and Pen/Strep (ready-to-use mixture ofantibiotic penicillin and streptomycin (Roche Diagnostics GmbH DE);lyophilisate reconstituted in 20 ml; use of 2 ml per 1000 ml medium),

In order to get adhered to the plastic 300.000 PBMC per well werepreincubated overnight in 96 well flat bottom plates.

The next day tetanustoxoid (TT) (Chiron Behring) was added to the wellsin a final concentration of 2 to 5 μg/ml. The positive control wells(maximum proliferation/stimulation) only contained TT, to all otherwells antibodies (as purified IgG) were added in a final concentrationof 10 μg/ml. Murine Mab TAG-34 was included in the assay (finalconcentration 10 μg/ml). As nonstimulatory background control mediumalone was used. All assays were set up in triplicates.

After six days of further incubation (37° C., 5% CO₂, 95% humidity)³H-thymidine was added in a final concentration of 1 μCurie/ml and afteran additional incubation period of 16 h the plates were harvested andincorporated ³H-thymidine was determined in a beta-counter (FIG. 6).

Example 18 Cross-Reactivity of the OX40L Antibodies with Mouse OX40L

To determine the ability of the antibodies of the invention tocross-react with murine OX40L, serial diluted antibody and controlantibodies were incubated with K562-mOX40L cells, stably expressingmOX40L. Binding to K562 WT cells and K562-hOX40L cells, stablyexpressing hOX40L, was also assessed. As negative control a HuMabantibody directed against Keyhole Limpet Hemo-cyanin (alpha-KLH) wasused. Antibody RM134L, rat anti-mOX40L (eBioscience, San Diego, Calif.)was included as positive control for mOX40L expression. Antibody TAG-34,mouse anti-hOX40L (MBL, Nagoya, Japan) was included as positive controlfor hOX40L expression. For detection of bound human antibodies, afluorescein (FITC)-conjugated goat anti-human IgG antibody was used. Forthe detection of bound RM134L a biotinylated rabbit anti-rat IgGantibody (DAKO, Glostrup, Denmark) was used in combination withstreptavidin, conjugated with phycoerythrin (PE) (DAKO). For detectionof bound TAG-34, FITC-conjugated rabbit anti-mouse IgG antibody wasused. Calculations concerning EC₅₀ values or maximum binding at 20 μg/ml(Bmax) of the HuMabs tested were determined using non-linear regression(sigmoidal dose-response with variable slope) using Graphpad Prismsoftware.

Results: LC.001 according to the invention was able to bind to hOX40L asindicated by an EC₅₀ value of 5.16±2.93 μg/ml and a Bmax (MFI) value of385.22, but not to mOX40L or WT cells as shown by Bmax (MFI) values of11.41 and 9.67, respectively. Furthermore, LC.001(IgG4) according to theinvention was also able to efficiently bind to hOX40L as indicated by anEC₅₀ value of 8.19±1.05 μg/ml and a Bmax (MFI) value of 311.30, but notto mOX40L or WT cells as shown by a Bmax (MFI) value of 13.47 and 9.58,respectively. As expected, the negative control alpha-KLH did not bindto any cells. (FIG. 7). Therefore OX40L antibodies according to theinvention show at least 30 fold lower binding to mouse OX40L compared tohuman OX40L.

Example 19 Potential of OX40L HuMabs to Activate the Complement System

C1q and C3c Binding ELISA: To determine the ability of the antibodies ofthe invention to induce C1q binding and C3 activation, an ELISA platewas coated with serial diluted antibody and control antibodies. Asnegative control a human IgG4 (The Binding Site, Birmingham, England),that binds C1q very weakly, was used. Human IgG1 (The Binding Site) andalpha-KLH (IgG1) were included as positive controls. Subsequently,coated antibodies were incubated with recombinant C1q or human pooledserum, as a source of C3. For the detection of bound C1q, a rabbitantibody directed against C1q (DAKO) in combination with a swineanti-rabbit IgG antibody, conjugated with horseradish peroxidase (HRP)(DAKO) were used. For the detection of activated C3c (generated viaactivation of C3), a mouse anti-human C3c antibody (DAKO) in combinationwith a rabbit anti-mouse IgG antibody, conjugated with HRP (JacksonImmunoResearch Laboratories, West Grove, Pa.) were used. To assessdifferences in coating efficiencies, coated antibodies were visualizedwith a goat anti-human IgG antibody, conjugated with HRP. Calculationsconcerning EC₅₀ values or maximum binding at 10 μg/ml (Bmax) of theHuMabs tested were determined using non-linear regression (sigmoidaldose-response with variable slope) using Graphpad Prism software.

Results: LC.001 according to the invention was able to bind C1qefficiently as indicated by an EC₅₀ value of 2.1910.42 μg/ml and a Bmax(OD₄₀₅) value of 3.089. Furthermore, both positive control human IgG1and anti-KLH could bind C1q efficiently, as indicated by EC50 values of4.17±1.08 μg/ml and 2.57±1.51 μg/ml respectively, and Bmax (OD₄₀₅)values of 2.685 and 3.306 respectively. As expected, the negativecontrol human IgG4 did not bind C1q, as shown by an OD₄₀₅ Bmax value of0.353. Moreover, LC.001IgG4× according to the invention had lost thecapacity to bind C1q, as shown by an OD₄₀₅ Bmax value of 0.357.

In line with the C1q binding capacities, C3c deposition by LC.001occurred in an antibody-concentration dependent manner, with an EC50value of 2.67±0.16 μg/ml and a Bmax (OD₄₀₅) value of 2.614. Furthermore,both positive controls human IgG1 and anti-KLH could deposit C3cefficiently, as indicated by EC₅₀ values of 5.45±0.36 μg/ml and2.16±0.26 μg/ml respectively, and Bmax (OD₄₀₅) values of 2.543 and2.633, respectively. As expected, the negative control human IgG4 didnot deposit C3c, as shown by an OD₄₀₅ Bmax value of 0.095. Moreover,LC.001IgG4× according to the invention had lost the capacity to depositC3c, as shown by an OD₄₀₅ Bmax value of 0.090. (FIGS. 8 and 9).

Example 20 Potential of OX40L HuMabs to Bind to Fcγ receptors I, IIa andIIb

IgG-induced antibody-dependent cellular cytotoxicity (ADCC) is mediatedby Fcγ receptors (FcγR) on effector cells. To determine the ability ofthe antibodies of the invention to bind to FcγRs, IIA1.6 cells (derivedby limited dilution from IIA1 cells; Jones, B., et al., J. Immunol. 136(1986) 348-356) stably transfected with human FcγRI, FcγRIIa, FcγRIIband wild-type cells were incubated with serial diluted antibody andcontrol antibodies. As negative controls, human IgG2 (The Binding SiteLtd., UK), that does not bind FcγRI, and human IgG4 (The Binding Site),that does not bind FcγRII, were used. Human IgG1 (The Binding Site) wasincluded as positive control for FcγRI binding and human IgG3 (TheBinding Site) for FcγRII binding. Bound antibodies were detected by FACSanalysis using an antibody directed against human IgG conjugated withphyco-erythrin (PE). Calculations concerning EC₅₀ values or maximumbinding at 10 μg/ml (Bmax) of the HuMab tested were determined usingnonlinear regression curve fitting (variable slope) using Graphpad Prismsoftware.

LC.001 was able to bind to FcγRI efficiently (comparable to the controlIgG1 antibody) as indicated by an EC₅₀ value of 0.11±0.03 μg/ml and aBmax (MFI) value of 8041.54, but not to FcγRIIa and FcγRIIb as shown byBmax (MFI) values of 25.06 and 21.18, respectively.

LC.001IgG4× was less efficient in binding to FcγRI compared to LC.001and was comparable to the control IgG4 antibody, with an EC₅₀ value of0.86±0.12 μg/ml and a Bmax (MFI) value of 6030.07. No binding of LC.001IgG4× to FcγRIIa and FcγRIIb was observed (Bmax (MFI) values of 21.40and 19.27, respectively), whereas control IgG3 antibody was able to bind(Bmax (MFI) values of 536.65 and 418.59, respectively) (FIG. 10). TheEC₅₀ value for binding to FcγRI is therefore for LC.001IgG4× eight foldcompared to the EC₅₀ value of antibody LC.001.

Example 21 Potential of OX40L HuMabs to Bind to FcγRIIIa on NK Cells

To determine the ability of the antibodies of the invention to bind toFcγRIIIa (CD16) on Natural Killer (NK) cells, Peripheral BloodMononuclear Cells (PBMCs) were isolated and incubated with 20 μg/ml ofHuMab antibody and control antibodies in the presence or absence of 20μg/ml of a blocking mouse antibody to FcγRIIIa (anti-CD16, clone 3G8,RDI, Flanders, N.J.), to verify binding via FcγRIIIa. As negativecontrols, human IgG2 and IgG4 (The Binding Site), that do not bindFcγRIIIa, were used. Human IgG1 and IgG3 (The Binding Site) wereincluded as positive controls for FcγRIIIa binding. Bound antibodies onNK cells were detected by FACS analysis using a PE-labeled mouseanti-human CD56 (NK-cell surface marker) antibody (BD BiosciencesPharmingen, San Diego, Calif.) in combination with a FITC-labeled goatF(ab)₂ anti-human IgG (Fc) antibody (Protos immunoresearch, Burlingame,Calif.). Maximum binding at 20 μg/ml (Bmax) of the HuMab tested wasdetermined.

LC.001 was able to bind to FcγRIIIa efficiently (comparable to thecontrol IgG1 antibody) as indicated by a Bmax (MFI) value of 641.37.Addition of a blocking antibody against FcγRIIIa abolished binding ofLC.001 to NK cells (Bmax (MFI) value of 194.61 compared to backgroundstaining of 145.38). LC.001 IgG4× did not bind to FcγRIIIa and behavedcomparable to the control IgG4 antibody, with a Bmax (MFI) value of170.52, resulting in a Bmax of LC.001 IgG4× which is about only 10% ofBmax of LC.001. Addition of a blocking antibody against FcγRIIIa had noeffect on LC.001 IgG4× binding (Bmax (MFI) value of 174.26) (FIG. 11).

Example 22 Effect of hMab_hOX40L and Mab TAG-34 Binding to HUVEC(Primary Human Umbilical Vein Endothelial Cells/PromoCell)

Endothelial cells were described to express hOX40L (Kotani, A., et al.,Immunol. Lett. 84 (2002) 1-7). Human umbilical vein endothelial cells(HUVEC) naturally express hOX40L and therefore could be used as“endothelial cell model”. Aim of this assay was to determine the fate ofhOX40L on HUVEC cells after binding to antibodies TAG-34 and LC.001.

HUVEC were thawed and expanded in ECG-M media plus 2% FCS for 4 days. inT175-flasks (Sarstedt). The cells were plated into 24-well plates(10.000 cells/well).

After 3 days the media was changed to ECG-M+0.5% FCS. Addition ofantibody (<KLH> (antibody against Keyhole Limpet Hemocyanin), TAG-34 orLC.001 for induction of down-modulation) at 10 μg/ml and incubation for2,5 h or 24 h. Restaining of the HUVEC cells with TAG-34 or LC.001.FACS-staining with secondary antibody against murine IgG, labelled withAlexa488 (=<m>) or against human IgG, labelled with Alexa488 (=<h>) each10 μg/ml. FACS-measurement was done in FACS-scan (Becton Dickinson) andmean fluorescence intensity (MFI) was calculated.

The <KLH> antibody was used as unspecific, negative control.

Table 7 shows that addition of LC.001 does not result in down-modulationof OX40L expression on HUVEC cells neither after 2.5 nor after 24 h(compare line 4 with line 5 and 6). However addition of TAG 34 shows astrong (about 3-fold) down-modulation of hOX40L on HUVEC cells after 2.5h as well as after 24 h (compare line 10 with line 11 and 12).

Antibodies according to the invention in a concentration of 10 μg/ml donot induce downregulation of OX40L expression on HUVEC cells.

TABLE 7 Mab used for Mab used secondary Mab MFI downmodulation forstaining for FACS 2.5 h 24 h  1. media control — <h> 5.17 5.39  2. mediacontrol LC.001 <h> 28.52 24.99  3. <KLH> — <h> 4.76 4.74  4. <KLH>LC.001 <h> 31.44 23.07  5. LC.001 — <h> 36.52 30.78  6. LC.001 LC.001<h> 38.58 38.69  7. media-control — <m> 3.66 3.18  8. media-controlTAG-34 <m> 31.81 25.32  9. <KLH> — <m> 3.68 3.43 10. <KLH> TAG-34 <m>30.79 31.58 11. TAG-34 — <m> 9.44 7.39 12. TAG34 TAG-34 <m> 8.97 14.89

Example 23 Western Blot Analysis of TAG34, LC.001 and LC.005

40 and 100 ng hOx40L-His (R&D Systems, with a theoretical size of 28-34kDa) and the molecular weight marker Magik Mark XP (Invitrogen; 20, 30,40, 50, 60, 80, 100, 120, 220 kDa) were prepared forgel-electrophoresis. Therefore x μl Protein, 2.5 μl NuPage LDS (lithiumsalt of dodecyl sulfate) Sample Buffer (4×), 1 μl NuPage Reducing Agent(10×), and H₂O and 10 μl were put together and denatured for 10 min. at70° C. After that the samples were loaded onto a NuPage gel (Novex; 10%Bis-Tris) and run for 1 h at 150V in 1×MOPS Running Buffer (Novex).

The gel was blotted by a Semi-Dry-Blot onto a PVDF Membrane (Millipore;activation of the membrane by 5 min. incubation in methanol and 10 minincubation in 1×transfer buffer) using 1×NuPage Transfer Buffer(1×buffer, 0.1% Antioxidance, 10% methanol) for 1 h/50 mA in a semi drychamber. The membrane was blocked in 1×PBS/5% milk/0.5% Tween undershaking for 1 h at RT. The primary antibody (pAB) was diluted in1×PBS/1% milk/0.5% Tween, added and incubated overnight at 4° C.

LC.001: 1.9 μl (1.6 μg) in a total volume of 4 mlLC.005: 1.1 μl (1.6 μg)/4 mlTAG34: 1.6 μl (1.6 μg)/4 ml

The membrane was washed 3× for 10 min in 1× PBS/0.5% Tween. Thesecondary antibody (sAB) was diluted in 1×PBS/1% milk/0.5% Tween, addedand incubated for 1.5 h at RT. For LC.001 and LC.005 polyclonal antibodyagainst human IgG (Pierce) in a 1:10000 dilution was used as sAB; forTAG34 polyclonal antibody against mouse IgG from the Lumi-Light WesternBlotting Kit (Roche) in a 1:400 dilution was used as sAB. The membranewas washed 2× for 30 min with 1×PBS/0.5% Tween. For detection theLumi-Light Western Blotting Kit (Roche) according manufacturer'sinstruction was used. The results from the western blot were shown inFIG. 12. LC.001 is able to detect (dodecyl sulfate) denatured OX40Lwhereas LC.005 and TAG34 do not bind to denatured OX40L.

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1-38. (canceled)
 39. An antibody, characterized in that said antibodybinds OX40L, contains a Fc part derived from human origin, and does notbind complement factor C1q.
 40. The antibody according to claim 39,characterized in that the antibody does not bind to human Fcγ receptorson NK cells.
 41. The antibody according to claim 40, characterized inthat the antibody is a human antibody.
 42. The antibody according toclaim 40, characterized in that the antibody is a chimeric or humanizedantibody.
 43. The antibody according to claim 39, wherein the antibodybinds to OX40L with a K_(D) value of less than 10⁻⁸ M.
 44. The antibodyaccording to claim 43, wherein the K_(D) range is between about 10⁻¹² toabout 10⁻⁹ M.
 45. The antibody according to claim 44, characterized inthat the antibody is an antibody of human subclass IgG1, containing oneor more mutations selected from PVA236, GLPSS331 and/or L234A/L235A(numbering according to EU index).
 46. The antibody according to claim44, characterized in that the antibody is an antibody of human subclassIgG4.
 47. The antibody according to claim 46, characterized incontaining mutation S228P.
 48. The antibody according to claim 46,characterized in containing mutation L235E.
 49. The antibody accordingto claim 39, characterized in that does not activate complement factorC3.
 50. The antibody according to claim 39, characterized in that itdoes not elicit complement-dependent cytotoxicity (CDC).
 51. Theantibody according to claim 39, characterized in that it does not elicitantibody-dependent cellular cytotoxicity (ADCC).
 52. The antibodyaccording to claim 39, characterized in that it shows in an ELISA assayinhibition by blocking the interaction of immobilized OX40L with solubleOX40 at a coating concentration of 0.5 μg/ml OX40L with an IC50 value inthe range of 1 nM-4 nM.
 53. An antibody, characterized in that saidantibody binds OX40L and that the antibody comprises a variable regioncombination independently selected from the group consisting ofcombinations a) the light chain variable domain defined by amino acidsequence SEQ ID NO:1 and the heavy chain variable domain defined by SEQID NO:2; b) the light chain variable domain defined by amino acidsequence SEQ ID NO:3 and the heavy chain variable domain defined by SEQID NO:4; c) the light chain variable domain defined by amino acidsequence SEQ ID NO:5 and the heavy chain variable domain defined by SEQID NO:6; d) the light chain variable domain defined by amino acidsequence SEQ ID NO:7 and the heavy chain variable domain defined by SEQID NO:8; e) the light chain variable domain defined by amino acidsequence SEQ ID NO:9 and the heavy chain variable domain defined by SEQID NO:10; the light chain variable domain defined by amino acid sequenceSEQ ID NO:11 OR 16 and the heavy chain variable domain defined by SEQ IDNO:12.
 54. The antibody according to claim 39, characterized in that thehuman light chain variable region comprises an amino acid sequenceindependently selected from the group consisting of SEQ ID NO: 1, 3, 5,7, 9, 11 and
 16. 55. The antibody according to claim 54, characterizedin that the human heavy chain variable region comprises an amino acidsequence independently selected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10 and
 12. 56. The antibody according to claim 55,characterized in that the antibody comprises the light chain variabledomain defined by amino acid sequence SEQ ID NO:1 and the heavy chainvariable domain defined by SEQ ID NO:2.
 57. The antibody according toclaim 39, characterized in that the antibody comprises at least oneamino acid mutation in the Fc part causing non-binding to complementfactor C1q.
 58. The antibody according to claim 57, characterized inthat the antibody comprises a kappa light chain constant region asdefined by SEQ ID NO:13.
 59. The antibody according to claim 58,characterized in that the antibody comprises a constant region asdefined by SEQ ID NO:14 or SEQ ID NO:15.
 60. An antibody binding toOX40L, comprising a variable light chain and a variable heavy chain,characterized in that the variable heavy chain comprises CDR3 selectedfrom SEQ ID NOs: 26-29 and/or the variable light chain comprises CDR3selected from SEQ ID NOs: 40-45.
 61. The antibody according to claim 60,characterized in that the variable heavy chain comprises CDR1 selectedfrom SEQ ID NOs: 17-20 and CDR2 selected from SEQ ID NOs: 21-25 and/orthe variable light chain comprises CDR1 selected from SEQ ID NOs: 30-34and CDR2 selected from SEQ ID NOs: 35-39.
 62. An antibody, characterizedin that it is produced by a cell line selected from the group consistingof cell lines: hu-Mab<hOX40L>LC.001, hu-Mab<hOX40L>LC.005,hu-Mab<hOX40L>LC.010, hu-Mab<hOX40L>LC.019, hu-Mab<hOX40L>LC.029 andhu-Mab<hOX40L>LC.033.
 63. The antibody according to claim 39characterized in that the antibody is a Fab, F(ab′)₂ or a single-chainfragment.
 64. A method for producing an antibody according to claim 1,wherein said antibody is characterized in that the antibody binds toOX40L with a K_(D) value of less than 10⁻⁸ M and is modified in such amanner that said modified antibody does not bind complement factor C1qand/or human Fcγ receptor on NK cells, said method comprising: a)providing a host cell comprising an expression vector which comprises amodified first nucleic acid and a second nucleic acid encoding the lightchain of said antibody; b) culturing said host cell under conditionsthat allow synthesis of said antibody; and c) recovering said antibodyfrom said host cell.
 65. A nucleic acid molecule encoding an antibodymolecule of claim
 39. 66. A vector comprising the nucleic acid moleculeof claim
 65. 67. A host cell comprising the vector of claim 66.